Heteroptera: Plataspidae): a Recent Invader to the United States

Biology and Management of Megacopta cribraria (Fabricius) (Heteroptera: Plataspidae): A Recent Invader to The United States

Julian R. Golec

A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Masters of Science

Auburn, Alabama December 13, 2014

Key words: kudzu bug, overwintering biology, host plant, natural enemies

Approved By

Xing Ping Hu Professor of Entomology Joseph E. Eger, Jr. Research Scientist, Dow AgroSciences Arthur G. Appel Professor of Entomology and Interim Dean of Research David W. Held Associate Professor of Entomology

1 2

Abstract 4

Identified as both an agricultural pest of soybean and a residential nuisance pest, 6

Megacopta cribraria (F.) (Heteroptera: Plataspidae), is a recently invasive pest species to 7 the southeastern United States. Isolated to a few northeastern Georgia counties upon 8 initial detection in the fall of 2009, it has subsequently spread to 13 states where it can 9 now be found as far north as Washington D.C. and as far west as Arkansas. Most of the 10 research regarding this pest insect is focused on its impacts on agriculture and urban 11 environments, however its biology and physiology seem currently overlooked. 12

Information on these aspects could provide an important understanding of this insect in 13 its new habitat. Therefore investigations were completed on some biological and 14 physiological traits of M. cribraria that may provide an understanding of the rapid spread 15 and success of this insect in its newly exploited geographical range. Through laboratory 16 and field experiments, it was demonstrated that approximately 15% of females in three 17 overwintering populations in Lee Co. (Auburn, AL) had mated before entering winter 18 dormancy and sperm was stored in their spermatheca for up to seven months, oocytes in 19 mated overwintering females proceeded to post-blastoderm stage before the onset of 20 spring feeding/mating, all of the overwintering males had sperm in their testes, and the 21 ratio of females gradually increased in populations during overwintering. This study 22 indicates that both males and females are capable of reproductive quiescence. It also 23

suggests that pre-overwinter mate females, containing both eggs and sperm, may be able 24 to invade new territories and produce a subsequent generation in the absence of males; 25

and the spread of this insect may be attributed to founders effect. The ability of M. 26 cribraria to bypassing feeding on the presumed obligatory host, kudzu (Pueraria 27 montana (Lour.) Merr. variety lobata (Willd.) Maesen and S. Almeida), was also 28 addressed. Through no-choice greenhouse assays, overwintered generation M. cribraria 29 could bypass feeding on kudzu to oviposition on mung bean (Vigna radiata (L.) R. 30

Wilczek), soybean (Glycine max (L.) Merrill), butter bean (Phaseolus lunatus L.), and 31 black-eyed pea (Vigna unguiculata (L.) Walp). No significant differences were observed 32 in the number of egg masses, individual eggs, or in hatch rate among the five legume 33 species. First-generation M. cribraria developed on all legume species except black-eyed 34 pea, in approximately 55.6 d. Mortality from egg to adult was highest on black-eyed pea 35 and butter bean followed by kudzu, mung bean and soybean. Results of this study suggest 36 that kudzu is not an obligatory host for M. cribraria. Lastly, preliminary field surveys in 37 the summer 2013 and 2014 discovered a native Dipteran (Tachinidae) species 38

Strongygaster triangulifera utilizing adult M. cribraria for the development of its 39 offspring, while the non-native, but natural occurring, Paratelenomus saccharalis 40

(Hymenoptera: Platygastridae), was found in AL, GA, and MS. 41

iii

Acknowledgments 47 48

I am very grateful to my major professor, Dr. Xing Ping Hu, for her support, 49 guidance, patience, and endless encouragement that she has offered over the course of my 50 studies. I owe thanks to Dr. Joe Eger, an outside committee member, who has likewise 51 given me great support and input on both academic and personal affairs. I would also like 52 to thank my committee members, Drs. Arthur Appel and David Held, for their 53 willingness to assist me whenever possible, ongoing encouragement, and guidance. 54

I also owe a thank you to all the members of our lab group, but I especially want 55 to thank Liu Yang for her help with statistics, being good lab company, and for the day- 56 to-day operations, including greenhouse and lab maintenance, and field collections. 57

Additionally I would like to thank Matt, Dr. Nate Hardy, Yetz, Kunle, Femi, and 58 all the other member of Dr. Liu’s lab, whom I ‘bother’ on a daily basis. 59

Of course, I must thank my family for their support throughout this entire journey. 60

Table of Contents 68

69 Abstract ...... ii 70

Acknowledgments ...... iv 71

List of Tables ...... viii 72

List of Figures ...... ix 73

Chapter 1: General Introduction ...... 1 74

Biology of Megacopta cribraria ...... 1 75

Taxonomy ...... 1 76

External Morphology ...... 2 77

Reproductive Morphology ...... 4 78

Geographical Distribution ...... 6 79

Life cycle ...... 7 80

Seasonal Activity and Voltinism ...... 10 81

Plant Damage and Pest Status ...... 10 82

Economic Importance ...... 13 83

Natural Enemies and Predators ...... 15 84

Natural Enemies and Predators Abroad ...... 15 85

Rates of Parasitism Abroad ...... 16 86

Natural Enemies and Predators in the U.S...... 17 87

Control Strategies: Urban & Agricultural ...... 18 88

Urban Control Strategies ...... 18 89

Agricultural Control Strategies ...... 22 90

Objectives ...... 23 91

Chapter 2: Pre-wintering copulation and female ratio bias: life history characteristics 92 contributing to the invasiveness and rapid spread of Megacopta cribraria (Heteroptera: 93 Plataspidae) ...... 25 94

Abstract ...... 25 95

Introduction ...... 25 96

Materials and Methods ...... 29 97

Collection of insects ...... 29 98

Microscopic examination of male and female reproductive anatomy ...... 29 99

Data analysis ...... 30 100

Results ...... 32 101

Dissections of spermathecae and testes ...... 32 102

Egg development in females ...... 34 103

Sex ratios ...... 35 104

Discussion ...... 36 105

Pre-wintering copulation ...... 36 106

Sperm storage and egg development ...... 37 107

Sex Ratios ...... 39 108

Chapter 3: First-generation Megacopta cribraria (F.) (Heteroptera: Plataspidae) are not 109 restricted to the wild legume kudzu, Pueraria montant Lour. (Merr.) (Fables: Fabaceae) 110 ...... 41 111

Abstract ...... 41 112

Introduction ...... 42 113

Materials and Methods ...... 44 114

Plants and insects ...... 44 115

No-choice experiments ...... 45 116

Data analysis ...... 45 117

Results ...... 46 118

Oviposition ...... 46 119

Duration of instars ...... 47 120

Adults ...... 51 121

Mortality ...... 52 122

Discussion ...... 52 123

Chpater 4: Strongygaster triangulifera (Diptera: Tachinidae) as a parasitoid of adults of 124 the invasive Megacopta cribraria (Heteroptera: Plataspidae) in Alabama ...... 56 125

Chapter 5: Discovery of Paratelenomus saccharalis (Dodd) (Hymenoptera: 126 Platygastridae), an egg parasitoid of Megacopta cribraria F. (Hemiptera: Plataspidae) 127 in its expanded North American rage ...... 50 128

Chapter 6: Extension Publications ...... 66 129

Kudzu Bug Control In Residential Areas: Frequently Asked Questions (FAQ) ...... 67 130

Kudzu Bug Control In Soybeans: Frequently Asked Questions (FAQ) ...... 72 131

Conclusions and directions for further research ...... 76 132

References ...... 78 133

134

135

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138 139 140 141

vii

List of Tables 142 143 144 Table 3.1. Mean (± SE) numbers of egg masses and individual eggs, mean hatch rate, and 145 mean number of first, second, third, fourth and fifth instars, and adults. Due to low 146 survivorship in the three replicates of butter bean, raw numbers are given past the third 147 instar (indicated by asterisks [*]). Different letters within a column indicate a significant 148 difference at a P = 0.05 using Tukey’s HSD...... 50 149 150 Table 3.2. Pooled mean developmental days (± SE) of the egg stage and the five nymphal 151 instars (I-V) of M. cribraria on mung bean, butter bean (not included in third, fourth or 152 fifth instar, or adult analysis, indicated by asterisks [*]) black-eyed pea, soybean, and 153 kudzu...... 50 154 155 Table 6.2. Examples of common insecticidal products available at local retail garden 156 supply stores. Always read and follow all direction on the product label ...... 71 157 158 159 160 161 162 163 164 165 166 167 168 169 170 171 172 173 174 175 176 177 178 179 180 181 182

viii

List of Figures 183 184 185 Figure 1.1 Overall appearance of M. cribraria (Photo by J. E. Eger, Jr. 2010) ...... 3 186 187 Figure 1.2 Ventral view of female (left) and male (right) M. cribraria showing sternite 188 coloration and terminus shape ...... 4 189 190 Figure 1.3 Posterior-end view of the genital capsule of female (left) and male (right) ...... 4 191 192 Figure 1.4 Female reproductive tract. Abbreviation definitions; I-VII Ovarioles; PED: 193 pedicel; CAL: calyx; LOVD: lateral oviduct; COVD: common oviduct; GON: gonopore; 194 ACG: accessory gland; SPM: spermatheca ...... 5 195 196 Figure 1.5 Male testis. Testicular follicles represented by roman numerals I-VII; VD: vas 197 deferens; SPM: spermatozoa ...... 6 198 199 Figure 1.6 Instar development from first (far left) to fifth (far right) (Photo by Zhang et 200 al. 2012) ...... 9 201 202 Figure 1.7 Endosymbiont capsules indicated by arrows (Photo by J.E. Eger, Jr. 2010) 203 ...... 9 204 205 Figure 1.8 Feeding damage caused by M. cribraria on soybean (Photo by Seiter et al. 206 2013b) ...... 13 207 208 Figure 1.9 Kudzu bug aggregations on residential structures (Photo by D. R. Suiter 2009) 209 ...... 21 210 211 Figure 1.10 Staining of human skin (ankle) (Photo by D. R. Suiter 2012) ...... 21 212 213 Figure 2.1 Ruptured spermatheca containing sperm (indicated by arrow on left) and 214 spermatheca without sperm (right) (viewed under 100x) ...... 31 215 216 Figure 2.2 Sperm from male testes (viewed under 100x) ...... 31 217 218 Figure 2.3 Average monthly percentage (± SE) of females storing sperm before, during, 219 and after dormancy. The Chi-squared test indicated that the percent of females storing 220 sperm from Sept. 2013 to Mar. 2014 did not significantly differ (X2 = 1.135; df = 6; P = 221 .980). There was a significant difference when pre winter females (Sept. 2013) were 222 compared with 223

post-winter (End Mar. 2014) (X2= 10.796; df = 1; P = 0.001) females, indicating mating 224 had occurred. Asterisk (*) indicates significant difference (P < 0.005) ...... 33 225 226 Figure 2.4 Oocyte development: arrows indicate vitellogenesis (left) and blastoderm 227 formation (right) (pictures taken 02 Mar. 2013; viewed under 100x and 400x) ...... 35 228 229 Figure 2.5 Number and proportion of male and female M. cribraria collected per month. 230 Asterisks above a bar indicate significant differences in numbers of females and males 231 for that month (*, P<0.05; **, P<0.001) according to a Chi-squared test ...... 36 232 233 Figure 3.1 Mean number (±SE) of egg masses per host plant. Different letters on bars 234 indicate a significant difference at a P = 0.05 using Tukey’s HSD ...... 46 235 236 Figure 3.2 Mean number (± SE) of eggs per host plant. Different letters on bars indicate a 237 significant difference at a P = 0.05 using Tukey’s HSD test ...... 49 238 239 Figure. 3.3 Mean developmental days (± SE) of each instar reared on mung bean, 240 soybean, kudzu, butterbean, and black eye pea. Different letters on bars indicate a 241 significant difference at a P = 0.05 using Tukey’s HSD test ...... 49 242 243 Figure 3.4 Mean developmental days (± SE) from egg to adult M. cribraria. Different 244 letters on bars indicate a significant difference at a P = 0.05 using Tukey’s HSD test .... 51 245 246 Figure 3.5 Mean number (± SE) of M. cribraria developing to adult stadia. Different 247 letters on bars indicate a significant difference at a P = 0.05 using Tukey’s HSD test. ... 51 248 249 Figure 4.1 Strongygaster triangulifera larval instars removed from M. cribraria adults 250 (graduations on scale = 1 mm) ...... 57 251 252 Figure 4.2 Strongygaster triangulifera adult reared from M. cribraria adult ...... 58 253

Figure 5.1 Eggs of Megacopta cribraria parasitized (gray coloration) and unparasitized 254 (salmon coloration) by Paratelenomus saccharalis (Image by J. Golec, Auburn Univ.) 255 ...... 62 256 257 Figure 5.2 Paratelenomus saccharalis adult from lateral (A), dorsal (B), and frontal (C) 258 views (Images by E. Talamas, USDA-ARS) ...... 64 259 260 Figure 6.1. White panel trap used to catch adults (S. Horn and L. Hanula, 2011) ...... 70 261 262 Figure 6.2 Insecticide Efficacies ...... 75 263 264

CHAPTER 1 265 266 267 GENERAL INTRODUCTION 268 269 270 Biology of M. cribraria 271

Taxonomy 272 273

The family Plataspidae contains a reported 560 species and 59 genera (Davidova- 274

Vilimova and Stys 1980, Henry 2009). It is nested within superorder Pentatomoidea and 275 resides within the order Hemiptera. Megacopta cribraria was described by Fabricius in 276

1798 as Cimex cribrarius, and since several species have been placed in several genera, 277

Tetyra, Thyreocoris and Coptosoma, before being placed in the genus Megacopta 278

Coptosoma xanthochlora (Walker 1867) is a synonym. 279

Megacopta cribraria, commonly kudzu bug, was originally identified in the 280

United States by general and genital morphological studies (Eger et al. 2010, Ruberson et 281 al. 2010), followed by mitochondrial and nuclear DNA sequencing of the primary and 282 secondary endosymbionts (Jenkins and Eaton 2011) and genetic studies of the 283 cytochrome oxidase I sequence (Jenkins et al. 2011). Results of genetic testing matched 284 with M. cribraria and the closely related, M. punctatissima (Montandon), however, the 285 two species are likely conspecific (Eger et al. 2010, Hosokawa et al. 2014) and have been 286 considered synonymous (Yang 1934, Davidová-Vilímová 2006). 287

288

External Morphology 289 290

Morphological characteristics can be used to distinguish the Plataspidae from 291 other members of the Pentatomoidea of North America, and dimorphic characteristics 292 aide in the differentiation between male and female M. cribraria. One of the most 293 distinctive traits of this family is the complex folding of the hind-wing under the 294 scutellum that is evident in adult insects (Schuh and Slater 1995). However, to aide in the 295 identification to species level, Eger et al. (2010) provided a key to families of 296

Pentatomoidea in America north of Mexico that includes M. cribraria. The main 297 diagnostic characteristics used in the separation of Plataspidae, and thus M. cribraria (as 298 it is the only Plataspidae in the U.S.), from other Pentatomoidea are 1) two segmented 299 tarsi and 2) a greatly enlarged scutellum covering the majority of the abdomen. Overall, 300 the appearance of adult insects can be defined as such: 3.5- 6.0 mm in length, globular in 301 shape, and brown to olive green in color with dark punctuations along the dorsal side 302

(Figure 1.1) (Eger et al. 2010). 303

Dimorphic characteristics differ with respect to the coloration of the sternites, 304 curvature of the terminus, and shape of the genitalia capsule. The sternites of females are 305 lighter in color, but with an area of dark coloration restricted medially, while in males the 306 sternites are usually entirely dark in coloration (Figure 1.2). Females have a rounded 307 terminus, whereas males are truncated or blunt terminally. Lastly, the genital segments of 308 the male form a circular cup, unlike those of the female, which appear triangular in shape 309

(Figure 1.3). 310

As is common with other stinkbugs and most Heteroptera (Aldrich 1988), M. 311 cribraria contain scent glands used in the secretion of both defensive substances and 312

attractant pheromones (Kitamura et al. 1984). Nymphal M. cribraria contain dorsal 313 abdominal scent glands that function primarily for defensive purposes (Vilímová and 314

Kutalova 2011) and open through ostia located between tergal segments 3-4, 4-5, and 5-6 315

(Ahmad and Moizuddin 1975b, Schuh and Slater 1995). Adult M. cribraria will 316 additionally develop ventral metathoracic scent glands. 317

Secretions from the ventral metathoracic glands of adult Plataspidae are similar to 318 other pentatomid stinkbugs in that they are composed of alkanes and unsaturated 319 hydrocarbons. More specifically the secretion is a blend of undecane, dodecane, 320 tridecane, pentadecance, decenyl acetate, octenal, decenal, and oxo-hexenal (Baggini 321

1966, Kitamura 1984). 322

323

324

Figure 1.1. Overall appearance of M. cribraria (Photo by J. E. Eger, Jr. 2010). 325

326

327

Figure 1.2. Ventral view of female (left) and male (right) M. cribraria showing sternite 328

coloration and terminus shape. 329

330 Figure 1.3. Posterior view of the genital capsule of female (left) and male (right). 331

332 Reproductive Morphology 333 334

Relatively few studies have addressed the internal morphology of the kudzu bug 335

(Ahmad and Moizuddin 1975a). Moreover, only three studies have focused on the 336 internal reproductive structures of the family Plataspidae. Pendergrast (1957) conducted a 337 study of the reproductive organs of a single member of this family to better place 338

Plataspidae amongst other Heteroptera. Using only two species from the family 339

Plataspidae, Miyamoto (1957) determined the ovariole number of females. Ahmad and 340

Moizzuddin (1975a) provided the most extensive examination of the reproductive 341 morphology of both male and female M. cribraria. Descriptions of the reproductive tracts 342 of adults are contained within the report by Ahmad and Moizzuddin (1975a) and will not 343 be repeated here in detail. Briefly, however, the authors reported seven ovarioles in 344 females (Figure 1.4) and seven testicular follicles in males (Figure 1.5) in each of the 345 paired sexual organs. These findings are considered generalized features of lower 346

Pentatomoidea, including the Plataspidae and primitive pentatomids (Ahmad and Abbasi 347

1971). Also included in the generalized reproductive features of this family is the female 348 spermathecae morphology; e.g., having a highly sclerotized spermatheca, containing a 349 small pump region, lacking medial dilation in the spermathecal duct, and lacking 350 ectodermal sacs (Ahmad and Abbasi, 1971). 351

352

353

Figure 1.4. Female reproductive tract. Abbreviation definitions; I-VII Ovarioles; PED: 354

pedicel; CAL: calyx; LOVD: lateral oviduct; COVD: common oviduct; GON: gonopore; 355

ACG: accessory gland; SPM: spermatheca. 356

357

358

Figure 1.5. Male testis. Testicular follicles represented by roman numerals I-VII; VD: 359

vas deferens; SPM: spermatozoa. 360

361 Geographical Distribution 362 363

Megacopta spp. are native to the Old World and Oceania where they have been 364 reported from India, China, Australia, Indonesia, Japan, Korea, Macao, Myanmar, New 365

Caledonia, Pakistan, Sri Lanka, Thailand, Vietnam, Taiwan, and Malaysia (Montandon 366

1986, 1987, Distant 1902, Kirkaldy 1910, Matsumura 1910, Shroff 1920, Esaki 1926, 367

Hoffman 1931, 1935, Yang 1934, Ishihara 1937, Esaki and Ishihara 1950, Ahmad and 368

Moizuddin 1975, Hsiao and Ren 1977, Lal 1980, Ren 1984, Hirashima 1989, Easton and 369

Pun 1997). 370

Megacopta cribraria was first detected in the United States in Hoschton, Jackson 371

County, GA in October 2009, and represents the first report of a Plataspidae on the North 372

American continent (Eger et al. 2010), although the closely related Coptosoma 373 xanthogramma (White) (Hemiptera: Plataspidae) has been reported from the Hawaiian 374

Islands (Beardsley and Fluker 1967). Since the initial detection in 2009, it has 375 subsequently been identified in 13 U.S. states: Alabama, Arkansas, North Carolina, South 376

Carolina, Mississippi, Louisiana, Kentucky, Florida, Tennessee, Virginia, Delaware, 377

Maryland, and Washington D.C. (www.kudzubug.org). 378

379

Life cycle 380 381

Like all other Heteroptera, M. cribraria undergoes hemimetabolous development. 382

Beginning from an egg, it completes 5 nymphal instars before eclosing to an adult 383

(Figure 1.6) over a period of 25-56 days in its native regions (Eger et al. 2010). General 384 features of each instar from first-to-fifth can be seen in the coloration of the body, 385 scalloping of the lateral margin, increased number of setae, and development of the wing 386 pads (Moizuddin and Ahmad 1975). However a more reliable determination of the instar 387 is the width between the eyes and body size (Zhang et al. 2012). Adult longevity differs 388 between subsequent generations. In a bivoltine population of M. cribraria from China, 389

Zhang and Yu (2005) reported that first generation adults live 1.5-3 months, whereas the 390 second generation lives 9-10 months. These differences may be due to the overwintering 391 dormancy of the second-generation adults. 392

Males and females engage in large mating aggregations where females select their 393 mates before copulation (Hibino and Itô 1983, Hibino 1986, Hosokawa and Suzuki 1993, 394

1999, 2001). Pairs engage in copula by aligning in an end-to-end configuration and may 395 remain so in excess of 10 hours, even though sperm transfer is regularly completed 396 within 2-4 hours (Hoskawa and Suzuki 2001). This act of prolonged copulation is thought 397 to act as mate guarding by preventing re-mating of females by other males (Parker 1970, 398

Simmons and Siva-Jothy 1998). 399

Following copulation there is an incubation period of approximately 4-7 days 400

(Ahmad and Moizuddin 1977, Srinivasaperumal et al. 1992, Hosokawa and Suzuki 401

2001), after which females will deposit egg masses in parallel rows. Females prefer to 402 oviposit on actively growing leaf sheaths of the kudzu vine (Pueraria montana (Lour.) 403

Merr. variety lobata (Willd.) Maesen and S. M. Almeida) (Zhang et al. 2012). During egg 404 deposition, the female simultaneously places small, brown-colored endosymbiont 405 capsules underneath the egg mass. These contain two essential obligate bacterial 406 endosymbionts: the primary gamma-proteobacterial, Candidatus Ishikawaella capulata, 407 and the secondary alpha-proteobacterium, Wolbachia (Figure 1.7) (Fukatsu and 408

Hosokawa 2002, Hosokawa et al. 2007, Zhang et al. 2012). After a 3-5 day incubation 409 period, first instars hatch (Ahmad and Moizuddin 1977, Sprinivasaperumal et al. 1992) 410 and ingest the bacteria contained in the fecal capsules before dispersing to obtain plant 411 food. The acquisition of these bacteria is essential for proper development and 412 reproduction in the Plataspidae (Fukatsu and Hosokawa 2008, Hosokawa et al. 2010). 413

Without the mother-mediated transfer and ingestions of these symbiotic bacteria, 414

development of the insect is severely retarded and mortality increases dramatically 415

(Müller 1956, Hosokawa et al. 2007). 416

417

418

Figure 1.6. Instar development from first (left) to fifth (right) (Photo by Zhang et al. 419

2012). 420

421

422

Figure 1.7. Endosymbiont capsules indicated by arrows (Photo by J.E. Eger, Jr. 2010). 423

Seasonal Activity and Voltinism 424 425

In the United States, adult M. cribraria have three peaks of activity. The first peak 426 is seen after overwintering adults become active in late March through early May. The 427 second peak occurs after the first generation has developed, approximately late June to 428

August. The last peak is seen as adults migrate from feeding sites to overwintering sites 429 beginning in early October (Zhang et al. 2012). Nymphal development is gradual and 430 nymphal instars overlap with one another. First, second, third, and fourth instars can be 431 seen in development from late April through late June. Fifth instars are often seen late 432

June through July (Zhang et al. 2012). 433

Activity peaks of adults in the southeastern U.S. approximately correspond with 434 the activity of this insect in its Asian distribution (Tayutivutikul and Yano 1990, Wang et 435 al. 1996). While the seasonal activity of this insect occurs almost equally throughout its 436 range, its voltinism differs between newly invaded areas as compared to native regions. 437

One to three generations a year have been reported from eastern China (Wang et al. 438

1996), one generation a year has been observed in Southern Japan (Hibino and Itô 1983), 439 and two generations a year have been reported from south-central Japan and Thailand 440

(Tayutivutikul and Yano 1990). In warmer climates it has been indicated that M. 441 cribraria may be active throughout the entire year (Tippeswamy and Rajagopal 1998a). 442

In the U.S. it is reported as a bivoltine species (Zhang et al. 2012). 443

444

Plant Damage and Pest Status 445 446

The Plataspidae is one of few Hemipteran taxa that preferentially feed on legume 447 species (Schaefer 1988), and has thus been problematic to the production of legume crops 448 throughout the Old World (Yang 1934, Hasegawa 1965, Lal 1980, Ren 1984). The kudzu 449 bug prefers to feed on new plant growth, where it extracts photosynthate from the phloem 450

(Zhang et al. 2012). It can commonly be found feeding along plant stems, petioles, 451 leaves, pods, and possibly flowers (Zhang et al. 2012, Seiter et al. 2013b) of various 452 plants. Damage resulting from the feeding activity is seen as purple spots that later 453 coalesce to form large black necrotic regions (Figure. 1.8) (Thippeswamy and Rajagopal 454

2005), and extensive feeding may result in defoliation (Chaterjee 1934). As it is a phloem 455 feeder, M. cribraria produces copious amounts of honeydew that results in secondary 456 plant issues such as black sooty mold leading to reductions in photosynthetic ability of 457 the plant (Zhang et al. 2012). 458

The preferred host plants of M. cribraria in its native regions are the wild legume 459 kudzu, Pueraria montana (Lour.) Merr. variety lobata (Willd.) Maesen and S. M. 460

Almeida, and the cultivated soybean, Glycine max var. Merill (Ishihara 1950, Zhang et al. 461

2012), where it is most frequently reported as a pest of this bean in Asia (Hoffman 1932, 462

Ishihara 1950, Kobayashi 1981, Kono 1990, Wang et al. 1996, Wu and Xu 2002, 463

Hosokawa et al. 2007, Xing et al. 2008). In India it is commonly reported to attack 464

Dolichos lablab (L.) (Ahmad and Moizuddin 1975b, 1977; Thippeswamy and Rajagopal 465

1998, Thejaswi et al. 2008). However the bean plataspid is not limited to these species, as 466 it appears to have a broad host range of nearly 20 leguminous (Fabaceae) and 14 non- 467 legume species spanning 14 plant families in its native range (review by Eger et al. 468

2010). 469

Reports from the United States are limited on the host range of this insect in agro, 470 however it likewise feeds on kudzu and soybean. As M. cribraria is a recently invasive 471 species to the U.S., researchers are currently trying to determine whether or not it 472 similarly displays a broad host range. To date, Zhang et al. (2012) present the only field 473 choice experiments regarding this issue. They observed M. cribraria to feed on 10 forest 474 legume species and non-selectively oviposit on 8 species, although adults developed only 475 on soybean. While the development of the insect was apparently not possible on other 476 plants during the study, it indicates that M. cribraria is capable of utilizing additional 477 plant species for a nutrient source and can survive for an extend period of time on their 478 respective feeding plant. 479

Other studies such as Medal et al. (2013) conducted no-choice greenhouse 480 experiments and similarly found the plataspid to non-selectively oviposit on some of the 481 tested legume species. The authors found the plataspid to be capable of successful 482 development on the pigeon pea (Cajanus cajan L.), black-eyed pea (Vigna unguiculata 483

(L.) R. Wilczek), lima bean (Phaseolus lunatus L.), and pinto bean (Phaseolus vulgaris 484

L.) in addition to kudzu and soybean. 485

486

487

Figure 1.8. Feeding damage caused by M. cribraria on soybean (Photo by Seiter et al. 488

2013b). 489

490

Economic Importance 491 492

The kudzu bug is a well document pest of soybean throughout its native 493 distribution, effecting the production of this crop in India (Thippeswamy and Rajagopal 494

2005), China (Wang et al. 1996, Wu and Xu 2002, Xing et al. 2008), and Japan (Ishihara 495

1950, Kobayashi 1981, Takasu and Hirose 1985, Kono 1990, Hosokawa et al. 2007, 496

Kikuchi and Kobayashi 2010). Its impact on this bean can be extremely high. For 497 instance in China, researchers have reported yield reductions up to 50% (Wang et al. 498

1996). 499

In the cultivation of the lablab bean, Lablab purpureus (L.), a key edible bean 500 crop in India and the Middle East, M. cribraria has repeatedly been reported to occur at 501 high levels in fields throughout the cropping season (Ahmad and Moizuddin 1975, 502

Thippeswamy and Rajagopal 1998, 2005a; Rekha and Mallapur 2007, Thejaswi et al. 503

2008, Sujithra et al. 2008). This feeding activity has resulted between 9-44% reductions 504 in seed yield and size of this crop in India (Thippeswamy and Rajagopal 1998). 505

Similarly in the United States, the kudzu bug has begun to disrupt the cultivation 506 of the economically important soybean (Ruberson et al. 2012). In 2010, M. cribraria was 507 observed in soybean fields during early vegetative growth stages (V1-V3) in Georgia and 508

South Carolina (Seiter et al. 2013b). However no quantitative information on plant 509 impacts existed at that time. Limited studies have been conducted since then on the 510 effects of M. cribraria feeding, and have demonstrated that this insect is capable of 511 severely impacting soybeans. Preliminary studies by Greene et al. (2012) showed that M. 512 cribraria reduced crop yields in untreated fields upwards of 47%, however an average 513 yield loss was observed at 18%. Another study by Seiter et al. (2013b) demonstrated that 514 naturally occurring peak densities were capable of not only reducing yield by nearly 60%, 515 but feeding stress reduced seed weight, and effected pod size and seed-set per pod of 516 soybean. 517

In economic terms, 2011 marked the first year yield losses were reported from the 518 kudzu bug. According to a report by the Midsouth Entomologist in 2011 (Musser et al. 519

2012), a total of 31,349 bushels were lost per acre at cost of pesticide treatment plus loss 520 of yield at US$988,034.00 throughout the Southern U.S. Comparing reports from 2011 to 521

2012 (Musser et al. 2013), 2012 showed an additional 366,600 acres contaminated by this 522 insect, and a further 61,000 acres treated with insecticides. Overall, representing a 24-fold 523 increase in areas contaminated with the kudzu bug, and a 6.4-fold increase in acres 524 sprayed from 2011 to 2012. In Alabama, this represents an average loss plus cost of 525 control around US$28.50 per hectare (Musser et al. 2013). 526

As this insect appears to be expanding its range, these numbers will likely 527 increase over time. This is extremely alarming as soybeans are the second most abundant 528 crop grown in the U.S., with the first being corn (www.epa.gov). In total, the U.S. 529 produced over 3 billion bushels of soybeans in 2012, and exported nearly 45%, or 1.345 530 billion bushels internationally that same year. The total value of the crop in 2012 was 531

US$43 billion dollars (According to the American Soybean Association via 532 www.soystats.com). 533

534 535 Natural Enemies and Predators 536 537 538 Natural Enemies and Predators Abroad 539 540 541 In Asia and regions once endemic to the kudzu bug, there exists a large egg 542 parasitoid complex, minor predatory insects and arthropods, and a single fungus that all 543 may offer some control of populations of M. cribraria. This includes 7 reported egg 544 parasitic Hymenopteran species, 2 predatory Heteroptera, and 2 separate reports of an 545 arachnid and fungus. 546

The Hymenoptera species include, Ensarsiella boswelli (Hayat), Ablerus sp. 547

(Howard), Dirphys boswelli (Girault) (Aphelinidae), Paratelenomus saccharlis (Dodd) 548

(Platygastridae), Trissolcus sp. (Scelionidae), and Ooencyrtus nezarae (Ishii) 549

(Encyrtidae). These all appear widespread throughout the Old World (Ahmad and 550

Moizuddin 1976, Srinivasaperumal et al. 1992, Polaszek and Hayat 1996, Rajmohan and 551

Narendran 2001, Zhang et al. 2003). 552

The occurrence of additional natural enemies throughout areas native to the kudzu 553

bug include the Heteropteran predators Reduviius sp. (Reduviidae) and Antilochus 554 coqueberti (Fabricius) (Pyrrhocoridae) (Ahmad and Moizuddin 1976), the spider 555

Oxyopes shweta (Tikader) (Araneae: Oxyopidae) (Borah and Sarma 2009), and the 556 entomopathogenic fungus Beauveria bassiana (Borah and Dutta 2002) found infecting 557

31% in nymphs and 19% in adults sampled in Assam, India. 558

559 Rates of Parasitism Abroad 560 561

The least dominate of the egg parasitoids are the Trissolcus sp. (Hymenoptera: 562

Scelionidae) that co-exists with O. nezarae in China. Collection of these wasps in a 563 soybean field in China accounted for less than 2% of the total parasitoids in agro (Zhang 564 et al. 2003). Alberus sp. (Hymenoptera: Aphelinidae), a wasp from southern India, 565 appears more dominate, being discovered developing within nearly 60% of M. cribraria 566 eggs collected from a lablab bean field (Rajmohan and Narendran 2001). 567

The most successful and frequently reported parasitoids of M. cribraria in Asia 568 are Paratelenomus saccharalis (Hymenoptera: Platygastridae), a specialist of the family 569

Plataspidae, and Ooencyrtus nezarae (Hymenoptera: Encyrtidae), a generalist egg 570 parasitoid of numerous phytophagous Hemipterans. Parasitism by these wasp species can 571 be very high, for example Wall (1928, 1931) found that parasitism by P. saacharalis 572 occurred in 51% of eggs that were field-collected from June-August in Guangdong 573

Providence, China. In Japan, Takasu and Hirose (1986) found parasitism of M. 574 punctatissima (=cribraria) eggs between 43-100% in a kudzu patch. While parasitism by 575

P. saccharlis in southern India ranged from 4.3-20.6% depending on both the crop and 576 the month (Srinivasaperum et al. 1992). 577

578 Natural Enemies and Predators in the U.S. 579 580

Being previously restricted to the Eastern Hemisphere many natural enemies and 581 insect predators that may keep populations of the kudzu bug below economic thresholds 582 in Asia and elsewhere, have not been found in the newly invaded region of the southeast 583

United States. Nonetheless, there have been various observations and discoveries of 584 native natural enemies attacking the kudzu bug in the United States. 585

Through field observations and molecular gut-content analysis Ruberson et al. 586

(2013) and Greenstone et al. (2014) reported several existing generalist predators 587 including Euthyrhynchus floridanus (L.) and Podisus maculiventris (Say) (Hemiptera: 588

Pentatomidae), Geocoris uliginosus (Say) and G. punctipes (Say) (Hemiptera: 589

Geocoridae), Zelus renardii (Kolenati) (Hemiptera: Reduviidae), Hippodamia 590 convergens (Guérin-Méneville) (Coleoptera: Coccinellidae), Orius insidiosus (Say) 591

(Hemiptera: Anthocoridae), Chrysoperla rufilabris (Burmeister) (Neuroptera: 592

Chrysopidae), the spiders Oxyopes salticus (Hentz) and Peucetia viridans (Hentz) 593

(Araneae: Oxyopidae), the entomopathic fungus Beauveria bassiana (Balsamo) 594

Vuillemin, and Phasia robertsonii (Townsend) (Diptera: Tachinidae) found parasitizing a 595 single adult. Additionally surveys by Golec et al. (2013) detected the presence of the 596 adult parasitoid, Strongygaster triangulifera (Loew) (Diptera: Tachinidae), and 597 collaborative efforts of Auburn University (Auburn, AL), University of Georgia (Griffin, 598

GA), and Emory University (Atlanta, GA) detected the presence of the egg parasitoid, 599

Paratelenomus saccharalis (Gardner et al. 2013). 600

601

Control Strategies: Urban & Agricultural 602

Urban Control Strategies 603 604

The seasons that urban residents will most frequently encounter kudzu bugs are 605 during fall, when the insect seeks overwintering sites, or in spring when it emerges from 606 overwintering sites in search for food (Suiter et al. 2010a, 2010b). Due to its attraction to 607 light colors, especially white (Horn and Hanula 2011), M. cribraria frequently ends up on 608 or in homes (Figure. 1.9) and when populations are high, will often come in contact with 609 vehicles and people (Eger et al. 2010). When homeowners first reported seeing large 610 aggregations of this lesser known urban pest to professional pest management companies, 611 their response was the application of pyrethroid-based sprays to the outside of effected 612 homes (Suiter et al. 2010), even though efficacies of pyrethroids had not been tested in 613 residential settings at this time. Seiter et al. (2013a) addressed this fundamental question, 614 and are so far, the only researchers to have tested the efficacy of insecticides on 615 residential surface materials (including vinyl, metal, bare and painted wood, and brick). It 616 showed that multiple pyrethroid and pyrethroid-neonicotinoid compounds 617

(Thiamethoxam + λ-cyhalothrin, Bifenthrin, Acetamiprid + Bifenthrin, and Imdicacloprid 618

+ β-cyfluthrin) caused 100% mortality within twenty-four hours, yet efficacies of the 619 treatments were reduced when applied to more porous building materials such as wood 620 and brick. However as this was a controlled laboratory study, not much is understood on 621 the extent to which environmental factors like UV-light and rain, have on the degradation 622 of these chemicals (Konstantinou et al. 2001). It has since been recommended that broad- 623 spectrum insecticides not be applied to residential structures due to their potential toxicity 624 to residents and unintended environmental consequences (Ruberson et al. 2010, U.S. 625

Environmental Protection Agency [EPA] 2013). Nonetheless, these results have 626 broadened our understanding of the use of insecticides against this invasive pest, and may 627 increase the efficacy of which spot treatments are performed on public structures. 628

To date, the most safe and effective methods and primary means for preventing 629

M. cribraria in urban settings are a combination of integrated pest management 630 approaches (IPM), which include physical, cultural, chemical, and biological practices. 631

The most effective means of preventing entry of nuisance pests into homes is to seal 632 cracks and crevices (Appel 2003); this can be done by caulking window seals, replacing 633 screening, and/or placing steel wool inside ventilation chimneys or pipes (Suiter et al. 634

2010, Golec et al. 2014a). Aggregating adults may be power-washed off residential 635 structures (Golec et al. 2014a). It is advised not to crush adults that are inside of homes; 636 they will release a foul odor and they may stain fabrics and skin (Figure 1.10) (Suiter et 637 al. 2010), and cause irritation of the skin and eyes (Wong and Mak 2012). They should be 638 removed by using either a household vacuum with pantyhose secured around the vacuum 639 tube with a rubber band, or they can be vacuumed with a shop-vac where the canister is 640 filled with soap water (Golec et al. 2014a). 641

Adult M. cribraria may enter home gardens and ornamental plantings around 642 residential structures, infestations of this manner may be mitigated by washing of the 643 infested plant(s) with water, knockdown techniques (placing a bucket containing soapy 644 water under effect plant(s)), or deterring insects from gardens/homes by the use of white 645 cross-vane bucket traps as described by Horn and Hanula (2011). 646

Another approach to lessen problems associated with the kudzu bug is to treat 647 kudzu patches adjacent to residential areas. This can be done by one or two ways: directly 648

exterminating the insects via application of insecticides, or by removing kudzu patches in 649 nearby residential areas. The best time to treat kudzu patches with insecticides is in early 650 spring when insects form large feeding aggregations in kudzu patches. Many products are 651 available in common local retail garden centers, and the most efficient insecticides for 652 homeowner use are those containing pyrethroids. Popular amongst these retail stores are 653 the deltamethrin-based D-FenseTM (Control Solutions Incorporated [CSI], Pasadena, TX, 654

USA), and Delta DustTM (Bayer Environmental Science, Research Triangle Park, NC, 655

USA). The second method, removal of the kudzu patch(es), can be accomplished by both 656 summer and fall mowing of vines, or by applying foliar herbicides to the leaves. 657

Removing kudzu by mowing is an intensive process that must be repeated nearly weekly 658 during the growing season, for multiple years. When pruning the vines and overall 659 biomass, it must be completely defoliated and brought flush with the ground. Although 660 this is a long-term method of kudzu control, it is the most environmentally friendly 661 means of eradication (Enole and Loewstein 2014). 662

When adopting to treat with herbicides, it is recommended to first treat with a 663 foliar applied herbicide when leaves are fully expanded in late spring or early summer. 664

This should be followed up with a second application in late summer or early fall to 665 disrupt the growth of any secondary leaves or shoots following the first application. 666

Again, treatment with herbicides, as with mowing, should be carried out for multiple 667 years. Herbicides containing triclopyr amine or glyphosate, active ingredients seen in 668 common products such as Bayer Advanced Brush Killer Plus ConcentrateTM and 669

Roundup ProTM, are very effect against kudzu (Enole and Loewstein 2014). 670

671

672

673

Figure 1.9. M. cribraria aggregations on residential structures (Photo by D. R. Suiter 674

2009). 675

676

Figure 1.10. Staining of human skin (ankle) (Photo by D. R. Suiter 2012). 677

Agricultural Control Strategies 678 679

In China, M. cribraria can be successfully controlled by the application of broad- 680 spectrum insecticides such as methamidophos, chlorpyrifos, beta-cypermethrin, 681 deltamethrin, beta-cypermethrin, or sumicidin. Susceptibility to insecticidal treatments 682 differs between nymphs and adults, yet when targeted appropriately these chemicals offer 683 extremely high rates of control in soybeans (Wang et al. 1996, Zhang and Yu 2005). 684

During the 2010 and 2011 soybean growing seasons in Georgia, field insecticidal 685 trails were completed on a wide range of chemicals, this reveled those containing 686 bifenthrin, cyhalothrin, zeta-cypermethrin, carbaryl, or acephate offered more than 80% 687 control 2 to 5 days after their application. Moreover the trade name insecticides Hero, 688

Brigade, Karate+Orthene, Endigo, Brigadier, Discipline, and Sevin had greater or equal 689 to 90% control 2 to 5 days after treatment (Roberts and Whitaker 2012). The results of 690 these trials were used to create guidelines on managing this insect in soybean fields 691 throughout the southeast, and have been incorporated in the Alabama (2014) and 692

University of Georgia (2013) Pest Management Handbooks, which recommend the use of 693 nine different insecticides spanning three classes; pyrethroids, neonicotinoids, and 694 organophosphates. 695

Insecticide treatments should always be applied in response to adult and nymphal 696 infestations, and only when field populations of the kudzu bug exceed the economic 697 threshold of 1 nymph (Reisig and Bacheler 2012) or 5 adults per sweep (Roberts 2013) 698 across the whole field. Adult Infestations typically begin at field edges and insects will 699 move inward as the season progresses (Seiter et al. 2013b). As field edges are the first 700 areas contacted by migrating adults, boarder rows should be prioritized for insecticide 701

applications early in the season. Subsequent applications should focus on where the 702 insects have since penetrated. Chemicals offering longer residual times should be 703 considered as adults actively migrate from overwintering to feeding sites, which lasts 704 approximately 3 months (Zhang et al. 2012). 705

While the results from Roberts and Whitaker (2012) come from limited field trials 706

(Green et al. 2012) the selected chemistries appear to offer good control of the kudzu 707 bug. Chemical controls, however, often represent a quick solution to the problem and in 708 the case of the kudzu bug there is often a need for multiple insecticide applications during 709 the adult migratory season (Roberts and Whitaker 2012). This makes a strictly chemical 710 based approach uneconomical and unsustainable. Furthermore many of the recommended 711 trade name insecticides are broad spectrum in nature and may be toxic to beneficial 712 arthropods, thus inhibiting their role in regulating pest insect populations. For instance 713 the newly discovered P. saccharlis and S. triangulifera may be sensitive to current 714 insecticidal treatments as research completed on other members of the Platygastridae 715

(Hymenoptera) show sensitivity to delamethrin (Rauno et al. 2010), chlorpyrifos, and the 716 entomopathogenic fungus, Bacillus thuringeiensis var. kurstaki (DiPelTM) (Amaro 2013), 717 while fenoxycarb (Grenier and Plantevin 1990, 1991) appears to be toxic to Tachinidae 718

(Diptera). Therefore it is imperative that the use of insecticides be timed appropriately 719 with adult migration, which should be monitored carefully, and insecticides should be 720 used cautiously towards the end of May to allow for the successful establishment of 721 parasitoids in agro. 722

723

Objectives 724 725

The kudzu bug, Megacopta cribraria (F.) (Heteroptera: Plataspidae), native to 726

Asia, was first detected in the U.S. in Georgia during October of 2009. Since then it has 727 invaded 13 southeastern states and is spreading northward and westward at a rapid rate. 728

When first observed it was determined only as an urban nuisance, however this status has 729 been upgraded to that of a serious agricultural pest. Currently, scientific literature 730 concerning this bug in the U.S. does not address biological and physiological traits that 731 have enabled the rapid invasion and exponential population growth of this insect in its 732

North American distribution. Therefore, the immediate project has set out to examine 733 such traits. The long-term goal of this project is to gain a comprehensive understanding 734 of some biological and physiological aspects of this insect that will aide in the 735 development of IPM programs against the kudzu bug. This project consists of three 736 supporting objectives. 737

1. Investigate I) development of the reproductive system, II) sperm storage, III) 738

oocyte development, and IV) sex ratio of overwintering adults 739

2. Determine if kudzu is a necessary host plant for successful development, or if 740

feeding on this plant can be bypassed 741

3. Preliminary survey of natural enemies, their distribution, and potential as 742

biological control agents. 743

For the three objectives, there are two separate hypotheses. 744

1. Overwintering second-generation adults are fertile at these times; females will 745

contain stored sperm. 746

2. Kudzu bugs will be able to develop on multiple legume species without the 747

need to have a preliminary feeding period on kudzu 748

CHAPTER 2 749 750 751 PRE-OVERWINTERING COPULATION AND FEMALE RATIO BIAS: LIFE 752 HISTORY CHARACTERISTICS CONTRIBUTING TO THE INVASIVENESS 753 AND RAPID SPREAD OF MEGACOPTA CRIBRARIA (HETEROPTERA: 754 PLATASPIDAE) 755 756

Abstract 757 758 Pre-winter copulation and sperm storage in overwintering adult Megacopta cribraria (F.) 759 was examined. Microscopic examinations of the spermathecae and ovaries were made in 760 females and of the testes in males that were collected weekly before, during, and after 761 dormancy. The results indicated that approximately 15% of females mated before 762 entering winter dormancy and sperm was stored in their spermatheca for up to seven 763 months, oocytes in mated overwintering females proceeded to post-blastoderm stage 764 before the onset of spring feeding/mating, all of the overwintering males had sperm in 765 their testes, and the ratio of females gradually increased in populations during 766 overwintering. This study indicates that both males and females are capable of 767 reproductive quiescence. The biological significance of these life cycle aspects is 768 discussed from the viewpoints of invasiveness and adaptation. 769

Introduction 770 771

The kudzu bug, Megacopta cribraria (F.) (Heteroptera: Plataspidae), is native to Asia 772 and India (review provided by Eger et al. 2010). Many authors indicate that this insect is 773 an occasional to serious pest of many leguminous crops and vegetables in its native 774 distribution (Takasu and Hirose 1985, Chen et al. 2009). It is commonly reported as a key 775 pest of the soybean (Glycine max L.) in China and Japan (Takasu and Hirose 1985, Wu 776 and Xu 2002, Xing et al. 2006) as feeding by this insect significantly reduces crop yield 777

(Chen et al. 1996). 778

The kudzu bug was first discovered in the United States in Georgia in October 779

2009, when adults were found aggregating on residential homes in abundance apparently 780 seeking overwintering sites (Eger et al. 2010, Suiter et al. 2010). At that time it was 781 defined only as a nuisance pest, yet the following year kudzu bugs were subsequently 782 discovered infesting vegetative soybean fields in Georgia and South Carolina (Seiter et 783 al. 2013b). This quickly elevated its status from an invasive urban nuisance to that of a 784 serious economic pest of the soybean in the U.S. (Greene et al. 2012), and a concern for 785 international trade (Ruberson et al. 2013). The distribution of the kudzu bug has rapidly 786 expanded since its initial detection, and can now be found in 13 southeastern states as 787 well as Washington D.C. (http://www.kudzubug.org/distribution_map.cfm). 788

In its temperate Oriental distribution the kudzu bug completes one to three 789 generations a year (Hibino and Itô 1983, Tayutivuikul and Kusigemati 1992, Wu et al. 790

2006), whereas in more tropical areas it may be active year round (Thippeswamy and 791

Rajagopal 1998). In its U.S. range, the kudzu bug undergoes two generations per year 792

(Zhang et al. 2012). It has a hemimetabolous life cycle that consists of eggs, five nymphal 793 instars, and adults (Ramakrishna Ayyar 1913, Ahmad and Moizuddin 1977, 794

Tayutivutikul and Yano 1990). When temperatures increase in spring, overwintering 795 adults become active and colonize kudzu (Pueraria montana (Lour.) Merr. var. lobata 796

(Willd.)), where they aggregate, feed, mate, and lay eggs. The first generation nymphs 797 develop on kudzu, and adults may switch to additional host plants such as soybean, or 798 remain on kudzu to develop the second generation (Zhang et al. 2012). The first instars 799 ingests bacterial symbionts from capsules deposited beneath the egg mass by their 800 mother, and will spend a quiescent period near the egg mass before dispersing to obtain 801 plant food (Fukatsu and Hosokawa 2008). Kudzu bug nymphs and adults feed on plant 802 phloem by inserting their stylet into stems, leaves, petioles, pods, and possibly flowers of 803 host plants (Zhang et al. 2012, Seiter et al. 2013b). In the fall, adults prepare for 804 overwintering by migrating to areas where they aggregate and shelter from adverse 805 environmental conditions. Reported overwintering shelters include under tree bark, on or 806 in structures, and in or under ground litter adjacent to kudzu patches, or near soybean 807 fields. At this time they often become a nuisance pest as they commonly invade 808 residential structures and home gardens (Eger et al. 2010, Waldvogel and Alder 2012). 809

Although many legume and non-legume species are reported as host plants to this insect, 810 only kudzu, soybean, pigeon pea (Cajanus cajan L.), black-eyed pea (Vigna sinensis L.), 811 lima bean (Phaseolus lunatas L.), and pinto bean (Phaseolus vulgaris L.) are confirmed 812 as being the primary reproductive hosts in the southeastern United States (Zhang et al. 813

2012, Del Pozo-Valdivia and Reisig 2013, Ruberson et al. 2013, Medal et al. 2013) 814

Despite the studies and reports on various aspects of its biology and control 815 methods in the U.S., kudzu bug overwintering biology has not been studied in detail. To 816 date, there is limited knowledge of ecological and behavioral information during this life 817

history period. This includes adult insect overwintering locations as well as habitat use 818 and aggregation behaviors of overwintering adults (Eger et al. 2010, Zhang et al. 2012). 819

Zhang et al. (2012) reported a small peak of egg laying that occurred in October in 820

Georgia, yet this generation did not complete development. We also observed oviposition 821 on 21 November 2013 by females collected on 05 November 2013 from a residential area 822 when kept in screen-capped glass jars and held at room temperature. However a separate 823 collection of females restricted to identical screen-capped jars, but kept outside did not 824 lay eggs. These observations suggested that adults may be capable of pre-winter mating, 825 and that adults do not enter a strong cold-season diapause, but rather a reproductive 826 quiescence or dormancy that can be broken with a period of warmer cold-season weather. 827

Thus environmental stimuli that induce overwintering may prevent fertilization and 828 subsequent embryogenesis in females, allowing the insect to stay reproductively dormant 829 at these times. 830

To address the limited understanding of kudzu bug overwintering biology, this 831 study investigated whether or not pre-winter and early spring copulation commonly 832 occur, if sperm is stored in the female spermatheca and male testes throughout dormancy, 833 if oocyte development occurs in overwintering females, and to determine the ratio and 834 variation in overwintering survival between males and females. Previous literature has 835 been limited to descriptions of the morphology of adult reproductive systems 836

(Pendergrast 1957, Ahmad and Moizuddin 1975, Kim and Lee 1993), and has not 837 reported on sperm storage in M. cribraria, or the family Plataspidae, although this has 838 been documented in other Hemipterans (Koshiyama et al. 1996, 1997a, 1997b; Kott et al. 839

2000, Kobayashi and Osakabe 2008). While sex ratios of this species have been observed 840

previously, they have only been determined in post-winter populations (Hibinio and Itô 841

1983, Zhang et al. 2012, Seiter et al. 2013b). Finally, we discuss how our findings may 842 offer an explanation of the invasiveness and rapid range expansion of kudzu bug in the 843 southeastern United States. 844

845 Materials & Methods 846 847

Collection of Insects. Adult M. cribraria were collected at approximately weekly 848 intervals before, during, and after dormancy in Auburn, Alabama (Lee, Co.) from 849

September 2013 through March 2014 when adult activity resumed in full. Populations 850 were sampled at three locations, including a large patch of kudzu, two mature pecan trees 851

(Carya sp.), and an area of ground litter adjacent to an additional kudzu patch. Pre-and- 852 post dormant individuals were collected with a sweep net, while dormant insects were 853 collected by hand from cracks, crevices or beneath bark on the main trunk of pecan trees, 854 as well as beneath ground litter, and from fallen logs adjacent to the kudzu patches. Bugs 855 were placed in a glass jar (10.16 x 7.62 cm) and transported to the laboratory. Individuals 856 were counted and separated, and the sex ratio was determined. 857

858

Microscopic examinations of male and female reproductive anatomy. Individual 859 insects were pinned and submerged in a 1% sodium chloride solution contained in a wax- 860 lined dissection tray prior to dissection. Dissections were made using BRI surgical super 861 fine micro scissors (Biomedical Research Instruments, Inc. Silver Spring, MD) under a 862 stereomicroscope (Meiji model EMZ-TR, Japan). A transverse cut was made across the 863

scutellum and hemelytra exposing the tergal surface of the abdomen, which was then 864 excised to expose the internal anatomy. 865

Ovaries and spermathecae, or testes were removed and placed onto individual 866 microscope slides (25 x 75 mm. VWR, Radnor, PA) mounted with a 1% sodium chloride 867 solution to prevent desiccation before the addition of a cover slide (22 x 22 mm. Fisher 868

Scientific, Pittsburgh, PA). An optical microscope with a video-camera attachment (Meiji 869 model ML2000 microscope [Japan] with Hitachi D.S.P color video camera model VK- 870

C370, Japan) was used to observe development status of eggs in the ovaries, and presence 871 of sperm in the testes and spermathecae. The spermatheca is highly sclerotized and had to 872 be mechanically ruptured with pressure applied to the coverslip, and then viewed under 873 phase-contrast microscopy to identify the presence of sperm. To confirm presence or 874 absence of sperm within female spermathecae (Figure 2.1), contents of the testes (Figure 875

2.2) were compared to the expelled liquid of the spermathecae. Approximately 20 live 876 adult insects were selected by hand from the three collection locations and were dissected 877 each week. More specimens were examined for egg development during later weeks. In 878 total, 552 females and 476 males were dissected. 879

880

Data analysis. Percent of females storing sperm and percent with observable egg 881 development were pooled and analyzed on a monthly basis. Percentage data were 882 subjected to a Chi-squared test (α = 0.05) to determine if there were any significant 883 differences between the total numbers of females storing sperm, and females storing 884 sperm on a successive monthly basis, as this would indicate when mating resumed post- 885

winter (SPSS Inc.). The same statistical method was use to compare the monthly numbers 886 of collected males and females. 887

888

Figure 2.1. Ruptured spermatheca containing sperm (indicated by arrow on left) and 889

spermatheca without sperm (right) (viewed under 100x). 890

891

892

Figure 2.2. Sperm from male testes (viewed under 100x). 893

Results 894 895

Dissections of spermathecae and testes. Sperm that was expelled from both female 896 spermathecae (Figure 2.1) and male testes (Figure 2.2) appeared as long, thin lance-like 897 filaments. Sperm motility was observed in sperm that was expelled from both male and 898 female reproductive organs, however proportions of live/dead sperm could not be 899 assessed, and no differences could be distinguished between the head and tail regions, or 900 any other fine details of the sperm morphology. 901

The percentages of females storing sperm on a pooled monthly basis from 902

September 2013 through March 2014 are shown in Figure 2.3. Overall, 15% of females 903 stored sperm in their spermatheca during overwintering months, indicating that they had 904 mated before overwintering. The average percentage (varying between 9%-23% in 905 individual samples) of females storing increased gradually from pre-wintering (Sept. 906

13%), to preparatory overwintering months (Oct. and Nov. 14-15%), with minor 907 fluctuations during overwintering months (Dec. through early Mar., 13-17%), and finally 908 spiked post-overwintering by the end of March (36%). The X2 test showed no significant 909 difference between females storing sperm before entering winter (Sept., 2013) through 910 overwintering samples (middle Mar., 2014) (X2 = 1.135; df = 6; P = 0.980), indicating 911 that no copulation occurred during wintertime, and the adults were reproductively 912 dormant. The difference became strongly significant when pre-winter (Sept., 2013) 913 samples were compared to post-winter (end Mar., 2014) samples (X2= 10.976; df = 1; P = 914

0.001), indicating the occurrence of post-winter copulations. 915

All the dissected males had sperm in their testes, indicating that they were capable 916 of mating immediately in the spring. We were not able to differentiate mated from virgin 917 males. 918

919

45 *

15 Percent (%) female storing sperm (%) female Percent 10

0 Sept. 2013 Oct. 2013 Nov. 2013 Dec. 2013 Jan. 2014 Feb. 2014 Mar. 2014 End Mar. 920

Figure 2.3. Average monthly percentage (± SE) of females storing sperm before, during, 921

and after dormancy. The Chi-squared test indicated that the percent of females storing 922

sperm from Sept. 2013 to Mar. 2014 did not significantly differ (X2 = 1.135; df = 6; P = 923

0.980). There was a significant difference when pre winter females (Sept. 2013) were 924

compared with post-winter (End Mar. 2014) (X2= 10.796; df = 1; P = 0.001) (indicated 925

by brackets over bars) females, indicating mating had occurred. Asterisk (*) indicates 926

significant difference (P < 0.005). 927

928

Egg development in females. Female kudzu bugs have a pair of ovaries, each composed 929 of seven ovarioles branching from the oviduct (Figure 2.4). During winter, females had 930 little development in their ovarioles with no evidence of oocyte formation from 931

September 2013 through middle February 2014. Before the onset of post-winter mating, 932 or feeding in the spring, oocytes began developing as indicated by the formation of the 933 blastoderm and vitelloengensis (Figure 2.4). Developing oocytes in overwintering 934 females were first observed within the ovarioles on February 26, 2014, and continued 935 until the last collection date on March 27, 2014. By then, approximately 54% of the 936 previously mated females (n=13) contained eggs of later developmental stages that 937 appeared ready for fertilization. 938

Developing eggs were also observed in collections of females between February 939

26 and March 27, 2104, though at a much lower rate (18%), in females (n=79) that lacked 940 stored sperm in their spermatheca, indicating that the oocytes develop before fertilization. 941

The more developed oocytes in mated females may suggest that stored sperm promote 942 oocyte development. 943

944

945

Figure 2.4. Oocyte development: arrows indicate vitellogenesis (left) and blastoderm 946

formation (right) (pictures taken 02 Mar. 2013; viewed under 100x and 400x). 947

948

Sex ratio. The numbers of overwintering males and females are shown in Figure 2.5. The 949

M:F ratio was close to 1:1 before overwintering. In subsequent months, the ratio slightly 950 decreased and females were continually collected from field sites in greater quantity than 951 males, accounting for > 50% of the collections each month. The lowest male ratio was 952 observed in February 2014, a month that had 8 d of below freezing temperatures (Hu, 953

2014). The last collection at the end of March 2014 had a M:F ratio of 1:1.53. A X2 test 954 showed a significant difference in the overall numbers of male and female collected from 955

October 2013 to March 2014 (X2 = 136.116; df = 1; P <0.001). The difference in the male 956 and female numbers between pre-winter and onset of post-winter mating (X2 = 33.884; df 957

= 1; P <0.001) was highly significant. The M:F ratio decline during the overwintering 958 period suggests that winter survival of females was greater than that of males. 959

960

* ** ** ** ** 100% 90% 80% 251 70% 410 395 469 463 532 60% Female 50% Male 40% 30% 243 20% 343 277 332 302 260 10% 0% Oct. 2013 Nov. 2013 Dec. 2013 Jan. 2014 Feb. 2014 Mar. 2014 961

Figure 2.5. Number and proportion of male and female M. cribraria collected per month. 962

Asterisks above a bar indicate significant differences in numbers of females and males 963

for that month (*, P<0.05; **, P<0.001) according to a X2 test. 964

965

Discussion 966 967

Pre-wintering copulation. About 15% (9-23%) of female kudzu bugs had mated prior to 968 entering winter dormancy. The relatively consistent percentages during winter months 969 indicate there was no copulation at that time. Kudzu bug females copulated, received and 970 stored sperm in their reproductive tract. This finding helps explain the observations of 971

October egg laying prior to seeking overwintering sites (Zhang et al. 2012), and the 972

November egg laying of the field collected females that were transferred from an 973 overwintering aggregation to laboratory rearing conditions (authors observations). The 974 findings also corroborate our hypothesis that adults do not enter a strong cold-season 975

diapause, but rather a reproductive dormancy that can be broken with a period of warmer 976 cold-season weather. 977

Copulation prior to overwintering is not uncommon in insects that overwinter in 978 the adult stage (Denlinger 1985, Tauber et al. 1986). This behavior is particularly 979 common in Hemipterians, such as Orius spp. (Kobayashi and Osakabe 2008), various 980

Nabidae (Kott et al. 2000, Roth and Reinhardt 2003), and the stinkbugs Nezara viridula 981

(L.) and Menida scotti (Puton) (Kiritani et al. 1966, Koshiyama et al. 1994, 1997a, 982

1997b, Jones and Sullivan 1981). This study is the first report on pre-winter mating and 983 the overwintering of mated females in reproductive dormancy in the family Plataspidae, 984 and in particular M. cribraria. 985

986

Sperm storage and egg development. Pre-winter mated female kudzu bugs can 987 successfully overwinter with their ovaries at the pre-vitellogenic stage of development, 988 and can store viable sperm in their spermatheca for up to seven months. After winter 989 sperm retained in the female spermatheca appeared to be as active as sperm within male 990 testes after spring activation, suggesting that sperm remains viable after long cold 991 storage. The percentage of females storing sperm was consistent during the overwintering 992 period. Females that were mated pre-winter had observable oocyte development on 27 993

February 2014, which indicates that adult dormancy was terminated on or near that date. 994

Developing eggs in unmated females indicates that maturation of oocytes occurred prior 995 to fertilized, and independent of mating; however, mating may stimulate oocyte 996 development and subsequent oviposition of fertilized eggs, as is common in other 997

Heteropterans (Soares et al. 2011). The observations of egg development show that 998

females undergo a rapid post-overwintering reactivation period in which oocyte 999 development abruptly begins and mating resumes. It may additionally indicate that 1000 females are able to release and utilize stored sperm to fertilize eggs at a time pivotal for 1001 maximizing subsequent instar development and success in spring. 1002

Questions remain whether the previously mated females belong to the previous 1003 spring generation or the summer generation, whether or not they need to re-mate in 1004 spring to stimulate reproductive events after winter dormancy, or if the prolonged storage 1005 of up to seven months decreases the quality of the sperm and reproductive ability of 1006 females. Of the insects that overwinter as mated adults, many have a capacity to maintain 1007 viable sperm during winter for several months and use the stored sperm for successful 1008 fertilization of eggs after spring activation without re-mating prior to oviposition in 1009 spring, and without decreasing the reproductive ability of females (Taylor 1984, Kott et 1010 al. 2000, Ceryntgier et al. 2004, Kobayashi and Osakabe 2008, Leong et al. 2012, Awad 1011 et al. 2013). Future studies should explore the fecundity of pre-winter mated females vs. 1012 post-winter mated females, egg hatch rate, and sex ratio in this insect. 1013

Male reproductive physiology appeared to remain active throughout dormancy as 1014 all of the males dissected from September 2013 through March 2014 contained active 1015 sperm within their testes. While this is a rare occurrence in Heteroptera (Saulich and 1016

Musolin 2011) it has not gone unobserved. The male stinkbug, Menida scotti (Puton), 1017 likewise remains physiologically active throughout winter and may even be capable of 1018 copulating during these times (Koshiyama et al. 1993). However dormancy and 1019 reproductive physiology of male insects during diapause are much less understood and 1020 have received far less attention than in female insects (Pener 1992). Proportions of viable 1021

sperm in males and females and the ability of females to utilized stored sperm for 1022 fertilization were not determined through this study, therefore future studies should 1023 address these questions. 1024

1025

Sex ratios. The overwintering male-to-female sex ratio gradually decreased from ≈1:1 1026 before entering overwintering to 1:1.53 after overwintering, which was similar to the sex 1027 ratio of 1:1.84 observed by Zhang et al. (2012) in early spring flight interception traps. 1028

The largest proportions of females were obtained in samples from February 2014, the 1029 coldest month with 8 d below freezing point. In many insects including some stinkbugs, 1030 males donate nutrients in their ejaculate to females during pre-winter copulation. Such 1031 nutrients may increase female overwinter survival yet reduce the survival of mated males 1032

(Koshiyama et al. 1996, Shapiro 1982, Boggs and Gilbert 1979, Kobayashi and Osakabe 1033

2008, Leong et al. 2012, Zhu et al. 2013). It has additionally been speculated that pre- 1034 winter copulation may stimulate lipid accumulation and increase cold hardiness, or 1035 change behaviors in females to better survive winter conditions (Kobayashi and Osakabe 1036

2008, Zhu et al. 2013). In this study, there was no sufficient evidence indicating that the 1037 increasing female ratio was a result of higher survival of mated females because the 1038 increase in the percentage of mated vs. unmated females was not statistically significant; 1039 however, it may be explained as a cost of pre-winter copulation for males. Yet trade-offs 1040 such as nutrient contents, cold hardiness and longevity of mated and virgin males and 1041 females remain to be investigated. It is likely that the percentage of females mated prior 1042 to overwintering, the overwintering survival, the viability of sperm and its storage in 1043

female spermathecae, varies between years depending on the temperature and differences 1044 in microclimatic conditions. 1045

1046

In summary, our study demonstrates several previously unreported aspects of the life 1047 cycle of M. cribraria. These new findings may represent important mechanisms involved 1048 in the invasiveness and rapid spread of this species throughout the southeastern U.S. Pre- 1049 winter mated females potentially increase the invasion of new feeding habitats without 1050 the need to mate again in the spring, enabling them to begin spring migration earlier than 1051 unmated females and to be the first to exploit feeding hosts in new geographical 1052 locations. It is possible that like in many other insects, mating prior to overwintering is 1053 adaptive because sperm storage by females indicates they can disperse, lay fertilized 1054 eggs, and successfully colonize new habitats when there is no access to mates (Socha 1055

2010, Leong et al. 2012). This is especially important for kudzu bug adults that have 1056 greater dispersal capabilities and high female overwinter survival rates. Additionally, the 1057 smaller numbers of overwintering males available for copulation in spring may limit the 1058 accessibility of sperm for fertilization. Females that contain sperm from a previous 1059 autumn mating may be able to overcome this obstacle (Härding et al. 2008). Most 1060 importantly, this strategy indicates that females might be able to establish subsequent 1061 populations via founders effects, which would aide in understanding the rapid spread and 1062 invasiveness of this species. 1063

CHAPTER 3 1064 1065 1066 FIRST-GENERATION MEGACOPTA CRIBRARIA (F.) (HETEROPTERA: 1067 PLATASPIDAE) ARE NOT RESTRICTED TO THE WILD LEGUME KUDZU, 1068 PUERARIA MONTANT LOUR. (MERR.) (FABLES: FABACEAE) 1069 1070

1071

Abstract 1072

1073

The biology of M. cribraria (F.) is not well understood in its new North American distribution, 1074 especially its development on alternative developmental or reproductive host plants. Therefore 1075 we investigated whether overwintered M. cribraria could directly feed and oviposit on other 1076 legume species and subsequently complete the first-generation on these species in a no-choice 1077 greenhouse assay. Overwintered M. cribraria successfully oviposited on mung bean (Vigna 1078 radiata (L.) R. Wilczek), black-eyed pea (Vigna unguiculata (L.) Walp), butter bean (Phaseolus 1079 lunatus L.), soybean (Glycine max L. Merrill) and kudzu (Pueraria montana (Lour.) Merr. 1080 variety lobata (Willd.) Maesen and S. Almeida). There were no significant differences in the 1081 number of days to oviposition, number of eggs and egg masses deposited, or in the hatch rates on 1082 the different legume species. First-generation M. cribraria developed on all legume species 1083 except black-eyed pea in ≈ 55.6 d with little variation in developmental time of the instars. Mean 1084 developmental days of the first, second, and fourth instars were consistent ≈ 7.0 d, while the third 1085 and fifth instars took longest to develop (≈ 12.3 d). There were significantly more first instars 1086

molting to second instars on kudzu, mung bean, and soybean than on butter bean. Mortality from 1087 egg to adult was greatest on black-eyed pea (100%), followed by butter bean (90%), kudzu 1088

(68.33%), mung bean (55%), and soybean (45%). These results indicate that kudzu is not an 1089 obligatory host for M. cribraria. 1090

1091

Introduction 1092 1093

Megacopta cribraria (F.) is an invasive agricultural and residential pest species that was 1094 first detected in the United States in 2009 (Eger et al. 2010). Since then, it has quickly expanded 1095 its U.S. distribution and is now found in 13 southeastern states (http://www.kudzubug.org/dis- 1096 tribution_map.cfm). This herbivorous insect feeds on plant phloem by probing stems, petioles, 1097 leaves, and pods of various plant species (Eger et al. 2010, Zhang et al. 2012, Sieter et al. 2013). 1098

The preferred hosts of M. cribraria are kudzu, Pueraria montana (Lour.) Merr. variety lobata 1099

(Willd.) Maesen and S. Almeida, and soybean, Glycine max (L.) Merrill (Tayutivutikul and Yano 1100

1990, Li et al. 2001, Wang et al. 2004, Xing et al. 2008). It is considered a serious agricultural 1101 pest of soybeans in many Asian countries (Ishihara 1950, Wang et al. 1996, Wu and Xu 2002). 1102

Megacopta cribraria undergoes five nymphal instars lasting ≈ 24-56 d in its native range, before 1103 eclosing into the adult (Eger et al. 2010). 1104

In the southeastern U.S., M. cribraria is thought to complete two-generations per year 1105

(Zhang et al. 2012). Depending on location, overwintering adult M. cribraria may spend up to 6 1106 mo without food when sheltered in overwintering sites (authors observations), and become active 1107 once temperatures increase in spring. Overwintered adults will either stay in kudzu patches or 1108 disperse to alternative hosts (Zhang et al. 2012) such as soybean, where they can cause 1109 tremendous damage (Seiter et al. 2013). 1110

Overwintered adult M. cribraria feed on multiple leguminous and non-leguminous plants 1111

(Zhang et al. 2012, Hu and Carroll 2012), yet they display a restrictive reproductive host range 1112

(i.e. able to develop from egg to adult) which is limited to legumes, namely kudzu (Zhang et al. 1113

2012, Medal et al. 2013), soybean (Zhang et al. 2012, Del Pozo-Valdivia and Reisig 2013) 1114 pigeon pea, Cajanus cajen L., black-eyed pea, Vigna unguiculata L., lima bean, Phaseolus 1115 lunatus L., and pinto bean, Phaseolus vulgaris L. (Medal et al. 2013) in the southeastern U.S. 1116

This biological trait, a broad host range yet limited reproductive range, is common of many 1117 native and invasive Pentatomoidea (review by Panizzi 1997), and is most likely the result of 1118 differences in adult and nymphal characteristics such as mobility (Panizzi et al. 1980, Schumann 1119 and Todd 1982), nutritional demands (Kehat and Wyndham 1972, Kester and Smith 1984), and 1120 stylet morphologies of adults and nymphs (see Moizzuddin and Ahamd 1975). 1121

Because this insect overwinters in or near kudzu patches (Waldvogel and Alder 2012), 1122 which are usually available before cultivated soybean crops (authors observations), it was 1123 anecdotally thought that M. cribraria must initially feed on kudzu to gain essential nutrients 1124 before dispersing to other feeding and reproductive host plants. However Smith (2013) and 1125

Seiter et al. (2013) reported that overwintered M. cribraria could infest early-planted soybeans, 1126 and Del Pozo-Valdivia and Reisig (2013) suggested that the first-generation is able to bypass 1127 kudzu and develop on soybean. Previous studies have not, however, demonstrated in-full if 1128 kudzu is necessary in the diet of M. cribraria, since adults that produced eggs or nymphs for 1129 these studies had been reared on or initially fed kudzu (Zhang et al. 2012, Medal et al. 2013, Del 1130

Pozo-Valdivia and Reisig 2013). 1131

To investigate if first-generation M. cribraria can develop on alternative host plants in 1132 the absence of kudzu, a no-choice greenhouse assay was conducted utilizing starved 1133 overwintered adults before the onset of feeding in spring. 1134

1135 Materials and Methods 1136 1137

Plants and insects. Mung bean, Vigna radiata (L.) R. Wilczek, black-eyed pea, Vigna 1138 unguiculata (L.) Walp, (http://www.nuts.com, Cranford, NJ), butter bean, Phaseolus lunatus L., 1139

(Alabama farmers cooperative), and soybean (‘93Y92’, DuPont-Pioneer, Des Moines, IA) were 1140 grown from seed beginning 10 February 2014, while kudzu was grown from field collected root 1141 crowns (Lee Co., Auburn, AL). Seeds and root crowns were placed in 3.7 L black plastic pots 1142 containing Sunshine Pro Premium Potting Soil (SunGro Horticulture Canada Ltd., Seba Beach, 1143

AB). Plants were grown and maintained under natural light conditions, 23 ± 3oC, 50-70% RH, 1144 and provided with water as needed in a greenhouse on the Auburn University campus (Lee Co., 1145

Auburn, AL). Four plants of each legume species were grown. Overwintered adult M. cribraria 1146 were collected from tree bark and leaf-litter near a kudzu patch at Park Town Creek, Auburn, AL 1147

(Lee Co.) (32°36’32.80” N, 85°28’30.83” W), and an area of fragmented forest adjacent to Route 1148

14 near E. Glenn Ave. (Lee, Co., Auburn, AL) (32°36’32.80” N, 85°30’02.66” W) on 19 March 1149

2014, before migrating to feeding plants. 1150

1151

No-choice experiments. On 21 March 2014, one of each legume species was placed inside a 1152

24.5 x 24.5 x 63.0 cm cage (BugdormTM, BioQuip Products, Rancho Dominguez, CA) and 10 1153 pairs of field-collected adult M. cribraria (10 males: 10 females) were introduced to the cages. 1154

Adults were allowed to feed on their respective legume plant as needed, and were removed after 1155

25 d. Oviposition occurred ≈ 21.5 d after adult introduction. Plants were then inspected and the 1156 number of egg masses and individual eggs per mass were counted. Upon hatching neonate 1157 nymphs were allowed 24 h to ingest the endosymbiont capsule contents, at which time they 1158 displayed wandering behavior from the egg mass, and only 20 neonates were selected to remain 1159 on their respective host plant per replicate (n = 60 nymphs per replicate). Three of the same 1160 caged legume species were considered one replicate. Egg masses occurred on both the mesh cage 1161 material or on the plant itself. If instars were on the cage they were transferred to the plant leaves 1162 with a fine haired paintbrush (size 3/0, Royal Brush Mfg. Merrillville, IN). The hatch rate and 1163 days required for completion of each nymphal instar were recorded every 2 d. When plants 1164 reached maturity (every ≈ 20 d), the same plant species in a vegetative growth stage was placed 1165 inside the cage and nymphs were encouraged to wander to the new plant. Once the remaining 1166 nymphs moved to the vegetative plant, the mature plant was removed. At the completion of the 1167 experiment the total days required for development from egg to adult, and nymph to adult 1168 mortality were determined. On butter bean three replicates were used until the fourth instar, then 1169 the remaining instars (n= 9) were transferred, due to low survivorship in the three replicates, to a 1170 single caged butter bean for the duration of the experiment. Observational results are given past 1171 the fourth instar for this plant. 1172

1173

Data analysis. Data were subjected to an analysis of variance (ANOVA) (SPSS, Inc. v. 22, 1174

Chicago, IL), and means separated using Tukey’s Honestly Significant Difference (HSD) 1175 procedure (P = 0.05) when appropriate. A Chi-squared (α= 0.05) test was used to determine 1176 whether there were significant differences in total numbers of adults or in numbers of males and 1177

females produced by each legume species, and for rates of nymph to adult mortality on the five 1178 legume plants. 1179

1180 Results 1181 1182

Oviposition. In the no-choice assays, it took an average of 21.5 ± 0.41 d for oviposition to occur, 1183 and the duration from initial introduction until oviposition of adult M. cribraria did not differ 1184 significantly among the five legume species (df = 4, 10; F = 0.235; P = 0.912). There were no 1185 significant differences in the number of egg masses (df = 4, 10; F = 0.951; P = 0.474) (Figure 1186

3.1) or numbers of individual eggs (df = 4, 10; F = 0.647; P = 0.642) (Figure 3.2) laid on the five 1187 legume species. The duration of the egg stage (9.3 ± 0.21) did not differ significantly among the 1188 legume species (df = 4, 10; F = 1.170; P = 0.381) (Figure 3.3). 1189

1190

12 a a 10 a

8 a a 6

Mean number of egg masses of egg number Mean 2

0 Mung bean Black-eyed pea Butter bean Soybean Kudzu 1191 1192 Figure 3.1. Mean number (±SE) of egg masses per host plant. Different letters on bars indicate a 1193

significant difference at a P = 0.05 using Tukey’s HSD test. 1194

1195

100 a 90 a a 80 a 70 a 60 50 40 30 20 Mean number of eggs number Mean 10 0 Mung bean Black-eyed Butter bean Soybean Kudzu pea 1196 1197

Figure 3.2. Mean number (± SE) of eggs per host plant. Different letters on bars indicate a 1198

significant difference at a P = 0.05 using Tukey’s HSD test. 1199

1200

Duration of instars. Total hatch rate was greatest on soybean (68.2%) followed by kudzu 1201

(52.0%), butter bean (51.3%), black-eyed pea (33.6%), and mung bean (23.9%). However there 1202 were no significant differences in the hatch rates among the five legume species (df = 4, 10; F = 1203

0.761; P = 0.574) (Table 3.1). No first instar (n= 60) successfully molted to second instars on 1204 black-eyed pea. After restricting the number of neonates to 20 per plant (n= 60 per complete 1205 replication) the mean duration of the first instar nymph (6.3 ± 0.67 d) did not differ significantly 1206 among the other four legume species (df = 3, 8; F = 0.167; P = 0.916), nor was the duration of 1207 second instar (7.1 ± 0.23 d) significantly different (df = 3, 8; F = 2.867; P = 0.104). However 1208 there were significantly greater mean numbers of first instar nymphs molting to second instar 1209 nymphs on mung bean (19.0 ± 1.0), soybean (14.7 ± 3.0), and kudzu (19.6 ± 3.3) than on butter 1210 bean (5.3 ± 2.4) (df = 3, 8; F 11.139; P < 0.005) (Table 3.1). Mean developmental days of the 1211 third instar took significantly longer on butter bean (20.6 ± 0.67) than on mung bean (11.3 ± 1212

1.76), soybean (10.0 ± 1.15), and kudzu (10.0 ± 0.00) (df = 3, 8; F = 21.697; P < 0.005) and only 1213

5 third instars (10.0%) molted to the fourth instar nymphs on butter bean. However there were no 1214 significant differences in the mean number of third instar nymphs on mung bean, butter bean, 1215 soybean, or kudzu (df = 3, 8; F = 3.438; P = 0.72) (Table 3.1). There was no significant 1216 difference in mean developmental days for fourth instar nymphs on mung bean (8.0 ± 1.15 d), 1217 soybean (10.0 ± 2.0), and kudzu (5.0 ± 1.0) (df = 2, 6; F = 3.00; P = 0.125) or any significance in 1218 the mean number of fourth instars nymphs among the these three legume species (df = 2, 6 F = 1219

0.675, P = 0.544) (Table 3.1). Lastly, development of the fifth instar took significantly longer on 1220 kudzu (18.3 ± 1.0 d) than mung bean (11.0 ± 0.00 d), but development days on soybean (14.3 ± 1221

0.9 d) was not significantly different between kudzu and mung bean (df = 2, 6; F = 6.50; P = 1222

0.031) (Figure 3.3) (Table 3.1). Transition of M. cribraria from egg to adult was completed in 1223

49-55 d on mung bean, followed by kudzu (51-60 d) and soybean (55-59 d) (Fig. 4), averaging 1224

51-58 d among the three bean species (Table 3.2) Mean developmental time from egg to adult 1225 did not differ significantly among mung bean (52.3 ± 1.8), soybean (57.7 ± 1.3), and kudzu (56.7 1226

± 1.3) (df = 2, 6; F = 1.855; P = 0.236) (Figure 3.4). 1227

1228

25 b b

20 Mung bean ab Soybean 15 a a Kudzu a a a a a a a a Butter bean 10 a a a a a Mean developmental days developmental Mean a a a a a Black-eyed pea

0 egg-stage I II III IV V 1229

Figure 3.3. Mean development days (± SE) of each instar (I-V) reared on mung bean, soybean, 1230 kudzu, butter bean, and black-eyed pea. Different letters on bars indicate a significant difference 1231

at P = 0.05 using Tukey’s HSD test. 1232

1233

1234

1235

1236

1237

1238

1239

1240

1241

Table 3.1. Mean (± SE) numbers of egg masses and individual eggs, mean hatch rate, and mean 1242 number of first, second, third, fourth and fifth instars, and adults. Due to low survivorship in the 1243 three replicates of butter bean, raw numbers are given past the third instar (indicated by asterisks 1244

[*]). Different letters within a column indicate a significant difference at a P = 0.05 using 1245

Tukey’s HSD. 1246

1247

1248

Table 3.2. Pooled mean developmental days (± SE) of the egg stage and the five nymphal instars 1249

(I-V) of M. cribraria on mung bean, butter bean (not included in third, fourth, or fifth instars, or 1250 adult analysis, indicated by asterisks [*]) black-eyed pea, soybean, and kudzu. 1251

1252 1253 1254 1255

60 a a

56 a 54

adult adult 52

Mean developmental days to days to developmental Mean 48

46 Mung bean Soybean Kudzu 1256 1257

Figure 3.4. Mean developmental days (± SE) from egg to adult M. cribraria. Different letters on 1258

bars indicate a significant difference at a P = 0.05 using Tukey’s HSD test. 1259

1260

Adults. There were no significant differences in the mean number of adults that developed on 1261 kudzu (6.3 ± 3.5), soybean (11.0 ± 2.3), and mung bean (9.0 ± 5.7) (df = 2,6; F = 0.268; P = 1262

0.774) (Fig. 5) (Table 2) or the total number of M. cribraria adults reared on these legume plants 1263

(X2 = 3.747; df = 2; P = 0.154) (Figure. 3.5). There were no significant differences between the 1264 number of males or females produced on mung bean (X2 = 0.333; df = 1; P = 0.564), butter bean 1265

(X2= 0.200; df = 1; P = 0.655), soybean (X2= 0.030; df = 1; P = 0.862), and kudzu (X2= 1.316; df 1266

= 1; P = 0.251). 1267

1268

1269 16 a a 14 a 12 10 8 6 4 mean number of adults of adults number mean 2 0 Mung bean 51 Soybean kudzu

1270 Figure 3.5. Mean number (± SE) of M. cribraria developing to adult stadia. Different letters on 1271

bars indicate a significant difference at a P = 0.05 using Tukey’s HSD test. 1272

1273

Mortality. Pooled percent morality from nymph to adult was greatest on black-eyed pea (100%), 1274 followed by butter bean (90.0%), kudzu (68.33%), mung bean (55.0%), and soybean (45.0%). 1275

The overall Chi-squared test showed a significant difference (X2 = 29.905; df = 4; P < 0.01) in 1276 nymph to adult mortality among the legume species. Mortality was significantly greater on 1277 black-eyed pea than kudzu (X2= 6.095; df = 1; P = 0.014), mung bean (X2 = 13.065; df = 1; P < 1278

0.005), and soybean (X2 = 20.862; df = 1; P < 0.005), but did not differ from butter bean (X2 = 1279

0.526; df = 1; P = 0.468). On butter bean, mortality was significantly greater than on soybean (X2 1280

= 5.535; df = 1; P = 0.020), and mung bean (X2 = 8.448; df = 1; P = 0.004), but did not differ 1281 from kudzu (X2= 3.063; df =1; P = 0.080). Mortality on kudzu was only significantly greater than 1282 soybean (X2 = 4.681; df = 1; P = 0.030). Mortality on mung bean was not significantly different 1283 from soybean (X2= 1.000; df =1; P = 0.317). 1284

1285 Discussion 1286 1287

Previous experiments (Zhang et al. 2012, Medal et al. 2013, Del Pozo-Valdivia and 1288

Reisig 2013) have used adult M. cribraria that were either reared on kudzu and first instars 1289 transferred to selected legume species, or adults that were collected in the field after feeding on 1290 kudzu. However our study utilized overwintering M. cribraria that were starved and deprived of 1291 kudzu for approximately 6 mo before experimentation in no-choice greenhouse assays. 1292

Overwintered adult M. cribraria oviposited on all the caged legume species, including those that 1293 were not suitable alternative hosts for the first-generation, an observation that is consistent with 1294

reports from other researchers (Medal et al. 2013, Zhang et al. 2012) on M. cribraria. The hatch 1295 rate did not differ significantly on any of the five legume plants, indicating that there was no 1296 effect of host plant on this life-stage parameter. First-generation adults were able to develop on 1297 soybean, kudzu, and mung bean, and in one replication of butter bean. The successful 1298 development on mung bean represents a new host record in the U.S. 1299

The lack of development of adult M. cribraria on black-eyed pea, and the successful 1300 development on mung bean, contradicts those of Medal et al. (2013). They found that M. 1301 cribraria could be reared from egg to adult on black-eyed pea, but not on mung bean. However 1302 the result of our experiments agrees with Zhang et al. (2012) in that there was no adult 1303 development on black-eyed pea, yet they did not test mung bean. Differences in developmental 1304 success on different plants may be because Medal et al. (2013) used field-collected adults after 1305 the availability of kudzu, or variety of black-eyed pea used in their experiments. 1306

In its native Asian distribution, 24-56 d are needed for M. cribraria to transition from egg 1307 to adult on various host species (Eger et al. 2010). In this study, M. cribraria developed 5-9 d 1308 slower on soybean when compared to no-choice experiments by Del Pozo-Valdivia and Reisig 1309

(2013). This result is likely a difference in rearing conditions and soybean variety. 1310

Mortality was significantly greater on both black-eyed pea and butter bean suggesting 1311 that these two legume species are not suitable alternative reproductive hosts of M. cribraria. 1312

However, mung bean and soybean are adequate alternative reproductive hosts for M. cribraria, 1313 as mortality was lower on soybean then on mung bean and kudzu. Furthermore, soybean had a 1314 significantly lower mortality rate compared to kudzu, suggesting that soybean is a more suitable 1315 reproductive host for M. cribraria. The high rates of mortality (> 45.0%) observed in the no- 1316 choice experiments could have been induced by restricting nymphal M. cribraria to small cages 1317

preventing their access to shade or of declining host plant quality. Researchers have previously 1318 had difficulties rearing this insect in controlled environments (Zhang et al. 2012, Del Pozo- 1319

Valdivia and Reisig 2013). 1320

Mean developmental time of the first, second, and fourth instars were consistent among 1321 mung bean, soybean, and kudzu at ≈ 7.0 d for each instar, however the third and fifth instars 1322 were slower to develop than the aforementioned stadia, lasting ≈ 10.0 d and ≈ 14.6 d respectively 1323 on mung bean, soybean, and kudzu. Slower development of the fifth instar has been recorded 1324 previously (Tayutivutikul and Yano, 1990), however there are no previous reports of slower 1325 development of the third instar. During this instar, M. cribraria nymphs undergo a physiological 1326 change developing functional dorsal abdominal scent glands opening through ostia on tergites 3- 1327

4 and 4-5 (Moizuddin and Ahmad, 1975). A consequence of this morphological change may be 1328 the result of a trade-off between nutrient reallocation to favor the secondary development of 1329 defensive features over gaining body mass (Tollrian and Harvell 1999, Steiner and Pfeiffer 2007, 1330

Brönmark et al. 2012). 1331

Although mortality > 90%, a small number of M. cribraria nymphs (n= 5) were able to 1332 successfully develop into adults after 59 d in one replicate of butter bean. Noteworthy is the 1333 developmental time of the third instar, which took an average of 21 d to molt to fourth instars 1334

(Fig. 3). This result indicates that there is most likely a plant factor inhibiting or delaying 1335 nymphal development. This may be due to plant nutritional quality and elements like nitrogen, 1336 which are known to retard the development of immature insects (Matton 1980, Scriber and 1337

Slansky 1981). Research should further investigate what these plant factors are that significantly 1338 delayed the development of third instar M. cribraria. 1339

While this study was limited to no-choice greenhouse experiments, it indicates that kudzu 1340 is not an obligatory host for this insect. However feeding on kudzu and/or other supplementary 1341 plant species, or host switching from adult to nymph, may benefit the overall development of M. 1342 cribraria. For instance, supplemental feeding on legumes that are high in lipids, such as soybean, 1343 have been shown to increase starvation tolerance and reproductive fitness in the southern green 1344 stinkbug, N. viridula (L.) (Panizzi and Hirose 1995), additionally host switching from nymph to 1345 adult N. viridula has been indicated to increased fitness (Panizzi and Saraiva 1993). Perhaps M. 1346 cribraria displays similar benefits and/or trade-offs from this behavior, and subsequent 1347 investigations should address how host switching and supplementary feeding may affect certain 1348 biological and life history traits of M. cribraria. 1349

Previous reviews (Panizzi et al. 1997) have emphasized the importance that nutritional 1350 ecology plays in the life cycles of Pentatomoidea of agricultural concern. Few studies have 1351 examined the effects of host plant and reproductive host plant switching has between stages and 1352 on adults of subsequent generations. Further research should address these topics, since a better 1353 understanding of host and reproductive plant use, the sequence in which these plants are used, 1354 and the dispersal from which wild to cultivated crops takes place (Panizzi et al. 1997) could 1355 improve control methods against M. cribraria in the southeastern U.S. 1356

CHPATER 4 1357

1358 STRONGYGASTER TRIANGULIFERA (DIPTERA: TACHINIDAE) AS A PARASITOID 1359 OF ADULTS OF THE INVASIVE MEGACOPTA CRIBRARIA (HETEROPTERA: 1360 PLATASPIDAE) IN ALABAMA 1361 1362 1363 The kudzu bug, Megacopta cribraria (F.) (Heteroptera: Plataspidae), also known as the 1364 kudzu bug, is native to Asia. Before its initial detection in the U.S. in October 2009 (Eger et al. 1365

2010), species of the family Plataspidae were not known in the Western Hemisphere. In its 1366 native range, M. cribraria is not regarded as a significant pest (Ruberson et al. 2013). However, 1367 in the U.S., its rapid dispersal, explosive population growth, and severe damage to soybean and 1368 other legume crops, as well as its offensive odor, has elevated its status from an urban nuisance 1369 to a serious legume crop pest throughout the Southeast. Adults and nymphs extract plant 1370 nutrients from the vascular tissues of host plants. Whereas kudzu, Pueraria montana Lour. 1371

(Merr.) variety lobata (Willd.), and soybeans, Glycine max (L.) Merr., are its preferred hosts, 1372 adults have been collected from numerous legume and non-legume plants (Gardner et al. 2013). 1373

This can be largely attributed to its adaptability, high mobility, the relative lack of natural 1374 enemies, availability of reproductive host plants and feeding hosts, as well as a lack of fully- 1375 developed and tested management strategies. As of 19 August 2013, it was confirmed in 11 1376 states and all 67 counties in Alabama (unpubl. data, W.A. Gardner, http://www.kudzubug.org). 1377

On 10, 13, and 18 July 2013, the authors collected via beat sampling approx. 300 M. 1378 cribraria adults from early-planted (01 April 2013 planting date) soybeans on the campus 1379

of Auburn Univ., Auburn (Lee Co.), AL (32°35’23.34 N 85°29’19.01 W). These overwintered 1380 adults were collected to determine female reproductive development; however, dissections of the 1381 collected adults under a stereomicroscope (Meiji EMZ-TR, Meiji Techno, Japan) revealed a 1382 solitary endoparasitoid dipteran larva. The authors observed dipteran larvae at various 1383 developmental stages (1st to 3rd instar) in the abdomen near the reproductive structures. Larvae 1384 appeared to affect oocyte development in M. cribraria females, and both males and females 1385 showed tissue damage proximate to the reproductive structures. Feeding also resulted in damage 1386 to the adult’s circulatory system evident by hemolymph inside the abdominal cavity. 1387

In total, 214 (107 F; 107 M) adult M. cribraria were dissected. The overall rate of 1388 parasitism was 5.14% regardless of gender, with the parasitism rate in females of 9.34%, which 1389 was 10X greater than the parasitism rate (0.93%) in males. The size of the various larval instars 1390 observed ranged from 0.7 mm to 3.5 mm in length, with the 3.5 mm length being the oldest stage 1391

(3rd instar) (Figure 4.1). 1392

1393

1394

Figure 4.1. Strongygaster triangulifera larval instars removed from M. cribraria adults 1395

(graduations on scale = 1 mm). 1396

The remainder of the collection (n = 86) was maintained at room temperature (25°C, 65% 1397

RH) in glass jars and provided with tender soybean stems and leaves. The transparent glass jars 1398 allowed for observation of the 3rd -instar larvae exiting the host body to pupate on the bottom of 1399 the jars or attached to the underside of soybean leaves. Adult tachinid flies emerged 10 d after 1400 pupation and ranged from 4.3-5.0 mm (mean: 4.66, SE: 0.09; n = 7) in body length (Figure 4.2). 1401

Adults were identified as Strongygaster (= Hyalomyodes) triangulifera (Loew) 1863 1402

(Diptera: Tachinidae: Tachininae: Strongygastrini). Specimens were deposited in the Insect 1403

Museum of Auburn University (voucher #116 - 2013) and ARS museum (voucher # USNM ENT 1404

00,039,875). 1405

1406

1407

Figure 4.2. Strongygaster triangulifera adult reared from M. cribraria adult. 1408

1409

Strongygaster triangulifera is a small parasitic fly that is widely distributed in North 1410

America (Reeves and O’Hara 2004). This New World fly typically parasitizes only adult insects, 1411

with a few reports of larval parasitism viewed as abnormal occurrences (Thompson 1954). It is 1412 most frequently reported as a parasitoid of adult Coleoptera with host species in more than 10 1413 families (Guimarães 1978, Gerding and Figueroa 1989, Purrinton et al. 1990, Reeves and O’Hara 1414

2004), but it is also reported as having a broad host range including representatives of 1415

Dermaptera, Hemiptera, Lepidoptera, Orthoptera (Arnaud 1978, Kevan et al. 1988), 1416

Hymenoptera (Shima 1999), and Pentatomidae of Heteroptera (McPherson et al. 1982). 1417

This is the second report of parasitism by an indigenous North American tachinid fly 1418 species of M. cribraria adults in its expanded U.S. range. Ruberson et al. (2013) reported 1419 recovery of the tachinid Phasia robertsonii (Townsend) from a single adult in 2012 in Tift Co., 1420

GA. Strongygaster triangulifera has several characteristics as a generalist parasitoid that warrant 1421 further evaluation for its utilization as a naturally occurring or augmented biocontrol agent in a 1422 management program of M. cribraria in the southeastern U.S. 1423

CHPATER 5 1424 1425 1426 DISCOVERY OF PARATELENOMUS SACCHARALIS (DODD) (HYMENOPTERA: 1427 PLATYGASTRIDAE), AN EGG PARASITOID OF MEGACOPTA CRIBRARIA F. 1428 (HEMIPTERA: PLATASPIDAE) IN ITS EXPANDED NORTH AMERICAN RANGE 1429 1430 1431

Megacopta cribraria F. (Hemiptera: Plataspidae), commonly known as the kudzu bug or 1432 bean plataspid, was first discovered in the Western Hemisphere in October 2009 (Eger et al. 1433

2010). The plataspid quickly spread from the 9 northeastern Georgia counties in which it was 1434 initially confirmed into 383 additional counties in Alabama, Florida, Georgia, Mississippi, North 1435

Carolina, South Carolina, Tennessee, and Virginia by the end of 2012 (Gardner et al. 2013). 1436

Subsequent reports show that the insect has now been confirmed in 4 additional states – 1437

Delaware, Kentucky, Louisiana, and Maryland – and the District of Columbia, bringing the total 1438 number of confirmed states to 12 (W.A. Gardner, unpubl. data). 1439

Ruberson et al. (2013) reported that several existing generalist predators and a single 1440 entomogenous pathogen had been recorded as attacking M. cribraria in its expanded range in the 1441 southeastern U.S. They also reported a tachinid, Phasia robertsonii (Townsend), parasitizing a 1442 single adult M. cribraria in 2012, but no parasitism of eggs or immatures was observed in their 1443

2010 and 2011 surveys in Georgia. Golec et al. (2013) discovered Strongygaster triangulifer 1444

(Loew) (Diptera: Tachnidae) parasitizing individual adults (mean parasitism = 5.14%; n = 214) 1445 collected from soybean, Glycine max (L.) Merr., in Auburn, AL, in April 2013. Ruberson et al. 1446

(2013) concluded that parasitism of M. cribraria eggs did not appear to be occurring in its 1447

expanded North American range based on the lack of egg parasitoids reported in their study as 1448 well as the lack of egg parasitism observed by Zhang et al. (2012) in their 2010 study in 1449 northeastern Georgia. 1450

Furthermore, research conducted at the USDA-ARS National Biological Control 1451

Laboratory in Stoneville, MS, confirmed that M. cribraria eggs were not attacked by 11 species 1452 of native egg parasitoids (8 from Pentatomidae and 1 each from Coreidae, Reduviidae, and 1453

Rhopalidae) plus 4 species of exotic egg parasitoids being assessed for the brown marmorated 1454 stink bug, Halyomorpha halys Stål, host range project (W.A. Jones, unpubl. data). 1455

On 30 May 2013, 2 M. cribraria egg masses with several abnormally dark-colored 1456 eggs were collected by Joni L. Blount (UGA) from kudzu, Pueraria montana Lour. (Merr.) 1457 variety lobata (Willd.), growing on the University of Georgia Campus in Griffin (Spalding Co.). 1458

Microscopic examination of the masses showed that some of the eggs were a reddish color, 1459 others were a charcoal gray color, and others were the normal salmon color. The egg masses 1460 were placed in a single No. 1 Gelatin Capsule (Eli Lilly and Company, Indianapolis, IN) and 1461 held at ambient room conditions. Two weeks later, 3 small hymenopterans were discovered in 1462 the gelatin capsule with the egg masses. Microscopic examination of the egg masses confirmed 1463 that the wasps had emerged from 3 of the previously discolored egg capsules. An egg mass of 22 1464 eggs collected 2 July 2013 from kudzu in the same vicinity exhibited the same coloration and 1465 subsequently yielded 18 parasitoids. 1466

In June 2013, egg masses oviposited by M. cribraria adults in soybean growing on the 1467

Auburn University Campus in Auburn (Lee Co.), AL, were observed with a gray coloration. On 1468

17 July 2013, 4 egg masses (mean number eggs per mass ± SD = 22.25 ± 1.89) were collected by 1469

Julian R. Golec (Auburn Univ.) at that location, placed in a Petri dish, and maintained at 25°C in 1470

an environmental chamber. Forty-eight wasps emerged from 3 of the 4 egg masses on 23 July 1471

2013 with parasitism rates of 95.4% (n = 22), 95.2% (n = 21), and 52.4% (n = 21). Eleven egg 1472 masses were subsequently collected from the same location on 5 August 2013, with all but 1 of 1473 those masses being parasitized (84.8% egg parasitism, overall). Microscopic examination of the 1474 parasitized eggs showed that the grayish coloration was caused by the either black coloration of 1475 the developing parasitoid within the cream-colored host egg capsule or discoloration of the lining 1476 of the egg capsule (Figure 5.1). This grayish-coloration is distinctive for parasitism and serves as 1477 an aid in identifying parasitized eggs in the field. 1478

1479

1480

Figure 5.1. Eggs of Megacopta cribraria parasitized (gray coloration) and unparasitized (salmon 1481

coloration) by Paratelenomus saccharalis (Image by J. Golec, Auburn Univ.). 1482

1483

Jannelle Couret (Emory Univ.) observed adult parasitoids under magnification probing 1484 and ovipositing into M. cribraria eggs collected from kudzu on and near the Emory University 1485 campus in Dekalb Co., GA (metropolitan Atlanta), in late June. Parasitized eggs were maintained 1486 in Petri dishes in environmental chambers. Adult parasitoids were later observed emerging from 1487 the egg masses and, specifically, from the gray-colored eggs. The gray eggs that did not hatch 1488 were dissected to find parasitoid larvae and adults that had not successfully emerged. 1489

Egg masses were collected from 5 locations in Dekalb Co. on 24 July 2013. These 1490 masses were transported to the Department of Entomology Laboratory on the UGA Griffin 1491

Campus where numbers of eggs per mass were counted and the masses were placed individually 1492 into 2-dram glass vials. The vials were stoppered with cotton and placed in an environmental 1493 chamber maintained at 28°C with 70 - 80% RH and on a 12:12 h photoperiod. Vials were 1494 examined periodically for parasitoid emergence. In total, 91 egg masses were collected from 1495 those 5 sites on that date with a mean (±SD) number of eggs per mass of 13.3 ± 5.1. Seventy- 1496 eight of those 91 masses (85.7%) were eventually confirmed as parasitized. Parasitism rates 1497 averaged 69.0% (n = 159), 77.9% (n = 144), 72.0% (n = 507), 66.7% (n = 310), and 55.9% (n = 1498

109) at the individual collection sites. 1499

Ten egg masses (21.2 ± 7.0 eggs per mass) were collected on 23 July 2013 from soybean 1500 growing on the UGA Bledsoe Research Farm in Pike Co., a county adjacent to Spalding Co. 1501 where the parasitoid was initially discovered. All but 1 of those masses was confirmed as 1502 parasitized with an overall 47.5% level of egg parasitism. 1503

Microscopic comparison of the adult parasitoids from the Dekalb Co. sites (Emory Univ.) 1504 with those from the UGA sites in Spalding and Pike counties indicated all were the same species. 1505

Specimens from these sites were identified by Walker A. Jones (USDA-ARS) as Paratelenomus 1506

saccharalis (Dodd) (Hymenoptera: Platygastridae) using the taxonomic key of Johnson (1996); 1507 they also appeared identical to specimens from a colony of P. saccharalis maintained in 1508 quarantine at the USDA-ARS National Biological Control Laboratory in Stoneville, MS. 1509

Voucher specimens from Georgia and Alabama sites were sent to Matthew L. Buffington and 1510

Elijah J. Talamas of the Systematic Entomology Laboratory at the National 1511

Museum of Natural History (Smithsonian Institute, Washington, DC); Talamas confirmed the 1512 identification as P. saccharalis using the key of Johnson (1996). Images accompanying 1513 confirmation of the identity were provided by E.J. Talamas (Figure 5.2). 1514

1515

1516

Figure 5.2. Paratelenomus saccharalis adult from lateral (A), dorsal (B), and frontal (C) views 1517

(Images by E. Talamas, USDA-ARS) 1518

1519

Paratelenomus saccharalis is widely distributed in the Eastern Hemisphere (Ruberson et 1520 al. 2013), but this is the first report of this parasitoid in the Western Hemisphere. Ruberson et al. 1521

(2013) further noted that known hosts of P. saccharalis are restricted to the family Plataspidae. 1522

The only known hosts of P. saccharalis were listed as M. cribraria, M. punctatissimum 1523

(Montandon), and Brachyplatys subaeneus Westwood in Asia, and Coptosoma scutellatum 1524

(Geoffrey) in Italy. Given this level of host specificity, P. saccharalis would not be expected to 1525 occur in the Western Hemisphere prior to 2009 because the first known occurrence of any 1526 members of the family Plataspidae in the New World was in October 2009 with the discovery of 1527

M. cribraria in Georgia (Eger et al. 2010). Yet, since these initial independent discoveries of the 1528 parasitoid in Alabama and Georgia, P. saccharalis has been confirmed parasitizing M. cribraria 1529 eggs in 17 additional counties in Georgia, 8 additional counties in Alabama, and 1 county in 1530

Mississippi (unpubl. data). 1531

Paratelenomus saccharalis was identified as a candidate for importation and release 1532 against M. cribraria in its expanded range in the U.S. because of its host specificity, wide 1533 geographic distribution in its native range, and its close biological association with M. cribraria 1534

(Ruberson et al. 2013). Walker A. Jones and Richard M. Evans have been assessing the host 1535 range of the parasitoid in quarantine (USDA-ARS National Biological Control Laboratory) for 1536 over 2 years and, based upon their data, prepared a petition to USDA-APHIS for release of the 1537 parasitoid. 1538

The introduction and sudden appearance of this parasitoid in the expanded M. cribraria 1539 range might never be determined. Its appearance over a wide geographic area and at relatively 1540

high parasitism rates soon after its discovery are also puzzling. It is highly likely that the 1541 establishment of the parasitoid occurred after M. cribraria was established. Perhaps parasitized 1542 eggs were introduced and even dispersed on shipments of plants or nursery stock or on fresh 1543 food products. Regardless, this is not the first report of an inexplicable appearance of an egg 1544 parasitoid following the accidental introduction of a plataspid. Beardsley and Fluker (1967) 1545 reported the establishment of Coptasoma xanthogramma (White) (Hemiptera: Plataspidae) in 1546

September 1965 as the first plataspid discovered in Hawaii. They also reported that no 1547 parasitoids and only few predators were attacking the introduced pest in its expanded range in 1548

Hawaii. Notes from the 1968 meeting of the Hawaiian Entomological Society (1969) indicate 1549 that a parasitoid, presumably Trissolcus sp. (Hynenoptera: Platygastridae), was found 1550 parasitizing C. xanthogramma eggs at several locations and on multiple islands in early 1968. 1551

Interestingly, the reported appearance of the parasitized eggs, percent parasitism, and the 1552 developmental timing of the parasitoid are consistent with those reported by Tagaki and 1553

Murakami (1997). 1554

CHPATER 6 1555 1556 1557 EXTENSION PUBLICATIONS 1558 1559 1560 ENTOMOLOGY SERIES 1561

TIMELY INFORMATION 1562

Agriculture & Natural 1563 1564

Kudzu Bug Control in Residential Areas: Frequently Asked Questions (FAQ) 1565 1566 1567 The kudzu bug is an invasive pest species from Asian and was first reported in Georgia in 2009. 1568

In late 2010 it spread to Alabama, and by the summer of 2014 it had been reported from all 67 1569 counties in the state. Since its accidental introduction it has been confirmed in more than 14 1570 southeastern states. First considered a nuisance pest in residential areas, it is posing a much 1571 greater threat than previously thought. In Asian countries, it has been a serious pest of soybean 1572 and vegetable beans. Here in the United States, it not only cause yield loss of soybeans, but also 1573 poses threats to international trade of agricultural products to Central America, and is an urban 1574 nuisance pest. Here are some of the most frequently asked questions concerning kudzu bug 1575 control around homes and in urban settings. 1576

Why are there so many kudzu bugs on my home in spring and fall? During the fall kudzu 1577 bugs will seek out protected areas to spend the colder winter months. In spring, the warm 1578

weather wakes the bugs up from their overwintering sites and they will begin flying in search of 1579 food plants. Additionally, they are attracted to light colors such as white and yellow. 1580

Unfortunately this means they may aggregate on or nearby your home, on you, or your vehicle, 1581 and may accidently wander into your home. March-April and September-October present the 1582 most common times that homeowners will encounter this insect. 1583

What should I do if kudzu bugs enter my home? Kudzu bugs can enter your home through 1584 various ways such as holes in window and door screening, or through gaps under doors. The best 1585 way to prevent this from happening is to secure your home; make sure that cracks/holes are well 1586 sealed, windows are tight, and any opening to the outside is screened. If insects do enter your 1587 home do not squish them, as they will emit a foul odor and can stain fabrics. 1588

! Get a vacuum cleaner and secure pantyhose to the vacuum tube with a rubber band, 1589

this will allow you to suck the bugs up and dispose of them. 1590

! If you have a shop vacuum you can fill the canister with hot soapy water and suck 1591

them up that way. 1592

What do they eat and why they are seen on many garden plants and yard ornamentals? 1593

Kudzu bugs are leguminous plant feeders. The primary hosts, or named reproductive hosts, are 1594 kudzu vines and soybeans. This means that they will have to feed on the primary hosts to be able 1595 to produce eggs. However they are capable of feeding on a wide range of leguminous and non- 1596 leguminous plants. This is particularly true in early spring and late fall when the preliminary 1597 hosts are unavailable but they are desperately in need of storing reserves for overwinter, or are 1598 hungry enough to non-selectively eat tender plants to obtain energy before host plants become 1599 available. 1600

Do they cause damage or kill garden vegetables, fruit trees, and landscape plants? Kudzu 1601 bugs suck out the sap of plants, and can cause severe yield loss to soybeans. They don’t chew 1602 holes or eat leaves. The effects of their feeding on vegetable beans and peas can range from 1603 discolored spots to the death of the plants. Their feeding on non-legume plants is often minor, 1604 and discolored spots are barely visible. 1605

How can I control kudzu bugs in my garden and outside my home? If kudzu bugs begin to 1606 show up outside your home or in you garden, chemical control is not recommended on home 1607 gardens or residential structures, due to the potential for run off and their toxicity. If bugs begin 1608 to aggregate on your home you may control them mechanically via: 1609

! Washing them off your home with a high-powered hose or soap-water. 1610

! In the fall, remove plant debris around your home and garden in order to prevent the 1611

bugs from overwintering near your home. 1612

! Eliminating nearby kudzu patches with the use of herbicides or mowing. 1613

! Place buckets of warm soapy water beneath garden plants and knockdown bugs from 1614

plants into the buckets, then dispose of them. 1615

! You may delay the planting of vegetable plants past the critical time of kudzu bug 1616

emergence. 1617

! Since these bugs are attracted to lighter colors, you may set up a white-panel trap on 1618

the edge of your property. Use a piece of white poster board and cut it in half, attach 1619

the two halves by cutting a line up the middle of the two piece and put them together. 1620

Place a bucket with soap-water underneath the panel trap and you may deter some 1621

bugs from landing on your home (Figure 6.1). 1622

1623

1624

1625

1626

1627

Figure 6.1. White panel trap used to catch adults 1628 (S. Horn and L. Hanula 2011). 1629

Do they bite or transfer disease? They don’t bite like mosquitoes; they don’t sting like wasps 1630 and bees. The most significant impact they have on humans is the pungent odor they discharge 1631 when disturbed. Your skin will be stained orange if you are in touch with the odor emission. 1632

They are not known transfer disease. 1633

Will killing kudzu around my house eliminate my kudzu bug problem? Controlling kudzu 1634 near your house will likely reduce the large number of kudzu bugs seen in spring, summer, and 1635 fall. This should be a community-wide effort to eradicate kudzu patches. However, kudzu bugs 1636 are strong fliers and regardless of whether or not there is kudzu near by, they may still end up on 1637 or near your home. 1638

Will treating Kudzu bugs with insecticides get rid of them? Spraying insecticides discourages 1639 these bugs temporarily, but does not provide lasting control. Large scales spraying of house 1640 siding or plants that are not in the bean family is not recommended and will not get rid of these 1641

insects. Kudzu bugs tend to migrate in spring and fall and infestations come in waves. Many 1642 frequent applications of insecticides, or other treatment methods will only help to reduce bugs 1643 currently in the area. More or less, the Kudzu bug is here to stay, we have to learn how to 1644 manage them come spring and fall. 1645

What insecticides can I use if I have to kill Kudzu bugs? If you treat kudzu bugs with 1646 chemicals, it is best to apply these directly to kudzu patches in the landscape. Any product using 1647 an active ingredient ending in –thrin should be effective against the kudzu bug. However, 1648 synthetic pyrethroids are toxic to bees and beneficial insects that naturally help to keep pest 1649 populations in balance. So timing of application is critical. Never use harmful chemicals on your 1650 home or garden vegetables. There are many insecticides labeled for kudzu bug control, but most 1651 are for agricultural crops not for home gardens. 1652

1653

Table 6.2. Examples of common insecticidal products available at local retail garden supply 1654 stores. Always read and follow all direction on the product label 1655

1656

1657

1658

1659

1660

1661

1662 1663 Kudzu Bug Control in Soybeans: Frequently Asked Questions (FAQ) 1664 1665 1666 1 2 1 3 Julian Golec , Timothy Reed , Liu Yang , and Xing Ping Hu 1667

1668 1 Graduate Student, Department of Entomology and Plant Pathology, Auburn, University 1669 1670 2Extension Specialist, Alabama Cooperative Extension Service, Auburn University 1671 1672 3Extension Specialist/Professor, Department of Entomology and Plant Pathology; Alabama 1673 Cooperative Extension Service, Auburn, University 1674 1675 First recognized as a nuisance pest when discovered in Georgia during the fall of 2009, the 1676 kudzu bug has become a serious pest of soybeans. Initial reports indicated that this invasive 1677 insect is capable of reducing soybean yields by nearly 60%. Yield loss due to kudzu bug feeding 1678 has been observed to reduce pod size, seeds per pod, and seed weight. Control of the kudzu bug 1679 in agricultural settings is possible and begins with proper insect identification, attention to 1680 economic thresholds, and timing of chemical treatments. As this insect begins to migrate to 1681 soybean in the coming the months, below are a few frequently asked questions concerning kudzu 1682 bug control in soybeans. 1683

Where do kudzu bugs tend to aggregate initially in soybean fields? When kudzu bugs first 1684 encounter a soybean field, they will aggregate abundantly on the outside rows, or edges of the 1685 field. As the season progresses they will move toward the center of the field. 1686

When will I find kudzu bugs in soybean fields? Adults have been observed entering soybean 1687 fields as early as May in South Carolina (V1-V3 growth stage). Once adult insects are observed 1688 in fields, they will persist there throughout the growing season. Nymphs are normally first 1689 encountered in June and last through September. 1690

What is the recommended scouting technique? Sweep netting (15” diameter net) or visual 1691 inspections of the crop are the proper scouting techniques. Make sure to sample the entire field 1692 and not just crop borders or edges. 1693

Do kudzu bugs reduce plant height when they infest seedling soybeans in large numbers? 1694

Yes, kudzu bugs feed on plant sap and thus reduce the amount of nutrients flowing through the 1695 plant. This will reduce the plants vigor. Additionally, high kudzu bug populations have been 1696 indicated to be able to reduce soy yield by 60%. However no damage to seeds has be seen. 1697

How will kudzu bug feeding affect soybean plants? Kudzu bugs feed by inserting their 1698 mouthparts into the plant stems, and they will suck the plant sap. This removes vital nutrients 1699 from the plants, reduces plant vigor, and can induce the growth of black sooty mold leading to 1700 various secondary plant problems. 1701

Are early season soybeans more susceptible to kudzu bug infestation? Field observations 1702 show that kudzu bug numbers tend to be greater in early-plant soybeans than in later planted 1703 soybeans. 1704

Do all pyrethroids provide at least 80% control shortly after application? Not all, but many 1705 pyrethroids provide high mortality after application, usually 2-5 days after treatment. Pyrethroids 1706 that initially give 80% control or better of kudzu bugs include products containing bifenthrin 1707

(Brigade, Discipline, Fanfare), gamma-cyhalothrin (Declare), lambda-cyhalothrin (Karate Zeon, 1708

Silencer), and zeta-cypermethrin (Mustang Max). Additionally, carbaryl (Sevin) and acephate 1709

(Orthene 97) also initially give better than 80% control of kudzu bugs (Fig.1). 1710

How and when should I treat my field? The Alabama Pest Management Handbook (2014) 1711 recommends treating fields before first bloom when kudzu bugs reach a density of 5 adults per 1712 plant across the entire field. The suggested treatment threshold once plants begin to bloom is 10 1713 adults per sweep net when sweeping across two rows, or when one nymph per sweep is 1714 collected. If immature kudzu bugs are easily and repeatedly found on petioles and main stems 1715 during visual inspections of the canopy, treatment is likely warranted. Border row applications of 1716 recommended insecticides have, in some cases, slowed the movement of the kudzu bugs into the 1717 center of fields. However, re-application of insecticides may be needed if applied before or 1718 during kudzu bug migration into soybean fields. Kudzu bugs continued to migrate into soybean 1719 fields at Prattville, AL through the third week of July in 2013. Make sure insecticide applications 1720 thoroughly penetrate the canopy as these insects feed on the stems and petioles of the plant. 1721

Are adults or nymphs more susceptible to chemical treatment? Both adults and nymphs are 1722 susceptible to insecticides. 1723

Will one application of an effective insecticide provide season long control when applied at 1724 least one month after soybeans emerge? No, these insects will migrate into soybean fields 1725 from other feeding sites, and their migration to fields may last as long as two-to-three months. 1726

You should only apply an insecticide in response to a kudzu bug infestation when the pest 1727 density reaches the economic threshold. When treating multiple times make sure to change the 1728 insecticide and the active ingredient to prevent insecticide resistance. 1729

When do population peak in soybean fields? Adult and nymphal peak are usually seen in 1730

September (corresponding to ~R5), while eggs tend to peak in August (~R2) 1731

1732 1733 1734

1735

Figure 6.2 Insecticide Efficacies 1736

CONCLUSIONS AND DIRECTIONS FOR FURTHER RESEARCH 1737 1738 1739 The work completed through this thesis has reported on several previously unknown 1740 aspects of the overwintering biology and physiology of the kudzu bug. It showed that both 1741 females and males stored sperm throughout winter dormancy, that both virgin and pre- 1742 overwinter mated females could develop oocytes post-dormancy before feeding, or in the case of 1743 virgin females, without having had received sperm, and that the there was significantly more 1744 females collected throughout the overwintering months. Furthermore these findings give support 1745 to those by Jenkins and Eaton (2011). They determined that the current population of M. 1746 cribraria in the U.S. was established from a single female lineage, thus indicating that the female 1747 who was responsible for the subsequent establishment of the insect throughout the southeastern 1748

U.S. was likely mated and storing sperm while in transit to North America. It was additionally 1749 determined through no-choice greenhouse assays, that the overwintering generation could feed 1750 and oviposit on alternative host plants, and the first generation could develop from egg to adult 1751 on soybean and mung bean. This suggesting that kudzu is not an obligate host plant for the kudzu 1752 bug. Furthermore, field surveys lead to the discovery of two locally existing parasitoids of the 1753 kudzu bug. While this research has helped to answer some fundamental questions about certain 1754 biological and physiological traits of the kudzu bug that may have aided in the rapid spread of 1755 this insect in the U.S., it has certainly created many other questions that the author hopes 1756 subsequent researchers will investigate. The author offers some suggestions for further research 1757 in the next paragraphs. 1758

In regards to the second chapter, some of the questions that remain currently unanswered 1759 are; what are the proportions of live-to-dead sperm within overwintering female spermathecae, 1760 does the proportion change throughout the course of the overwintering months, and does this 1761 prolonged storage of sperm have any effect on post-winter fecundity, hatch rate, or 1762 sex ratio compared to the spring and summer generations, and do females need to re-mate post- 1763 winter, or can they successfully utilize stored sperm to fertilize eggs. 1764

While it was determined that the overwintered generation kudzu bugs are able to develop 1765 on the alternative host plants, soybean and mung bean, indicating that kudzu is not necessary for 1766 reproductive development, they may in fact benefit from host switching. Adult kudzu bugs do 1767 demonstrate host switching in the field, which can be seen by overwintering adults feeding on 1768 various plants around or within kudzu patches just after emerging from dormancy (unpublished 1769 data available on some wild host species), then switching to kudzu when it becomes available, or 1770 switching from kudzu to soybean as the cultivated crop becomes available. Host switching is a 1771 particularly common feature of other Heteroptera of economic concern and have been indicated 1772 to increase insect fitness (see Panizzi et al. 1997). Future investigations should address such 1773 questions, in particular the effects, if any, of host switching between the adult to nymph or 1774 nymphal to adult from kudzu to soybean and vise-versa. 1775

1776 1777 1778 1779 1780 1781 1782 1783 1784 1785 1786 1787

1788 1789 1790 REFERENCES 1791 1792 1793

Ahmad, I., and M. Moizuddin. 1975. Some aspects of internal anatomy of Coptosoma 1794

cribrarium (Fabr.) (Pentatomoidea: Plataspidae) with reference to phylogeny. Folia. Biol. 1795

23: 53-61. 1796

Ahmad, I., and M. Moizuddin. 1975a. Some aspects of internal anatomy of Coptosoma 1797

cribraria (Fabr.) (Pentatomoidea: Plataspidae) with reference to Phylogeny. Folia Biolo. 1798

23: 45-49. 1799

Ahmad, I., and M. Moizuddin. 1975b. Scent apparatus morphology of bean plataspid 1800

Coptosma cribrarium (Fabricius) (Pentatomoidea: Plataspidae) with reference to 1801

phylogeny. Pakistan J. Zoo. 7: 45-49. 1802

Ahmad, I., and M. Moizuddin. 1976. Biological control measures of bean plataspids 1803

(Heteroptera: Pentatomoidea) in Pakistan. Proc. Entomol. Soc. Karachi. 6: 85–86. 1804

Ahmad, I., and M. Moizuddin. 1977. Quantitative life history of bean plataspid; Coptosoma 1805

cribrarium (Fabr.) (Heteroptera: Pentatomoidea). Pakistan J. Sci. Ind. Res. 20: 366- 370. 1806

Ahmad, I., and Q. A. Abbasi. 1971. Functional morphology and histology of the male and 1807

female reproductive organs of red pumpkin bug, Coridius janus (Fabr.) (Heteroptera: 1808

Dinidoridae) with its bearing on the phylogeny. Pakistan J. Zoo. 3:37-51. 1809

Alabama Pest Management Handbook. 2014. ANR- 2096. pp. 30, 258, 262. 1810

Aldrich, J. R. 1988. Chemical ecology of the Heteroptera. Annu. Rev. Entomol. 33: 211- 1811

38Amaro, J.T. 2013. Seletividade de productos biológicos aos parasitoides 1812

Trichogramma pretisum (Hymenoptera: Trichogrammatidae) e Telenomus remus 1813

(Hymentoptera: Platygastridae) em laboratório. Universidade Estadual de Londrina. 1814

Thesis. Abstract in English. 1815

Appel, A. G. 2003. IPM of occasional urban invader pest species. J. Entomol. Sci. 38: 151-158. 1816

Arnaud, P.H., Jr. 1978. A host-parasite catalog of North American Tachinidae (Diptera). 1817

USDA Misc. Publ. No. 1319. 1818

Awad, M., P. Kalushkov, T. Nedvedová, and O. Nedved. 2013. Fecundity and fertility of 1819

ladybird beetle Harmonia axyridis after prolonged cold storage. BioControl. 58: 657-666. 1820

Baggini, A. R., R. Bernardi, G. Casnati, M. Paven, and A. Ricca. 1996. Ricerche sulle 1821

secrezioni difensive di Insetti Emitteri Eterotteri. Rev. Espan. Entomol. 42: 7. 1822

Beardsley, J. W., Jr. and S. Fluker. 1967. Coptosoma xanthogramma (White) (Hemiptera: 1823

Plataspidae), a new pest of legumes in Hawaii. Proc. Hawaiian Entomol. Soc. 19: 367- 1824

372. 1825

Bin, F., and S. Colazza. 1986. Egg parasitoids, Hym. Scelionidae and Encyrtidae, associated 1826

with Hemiptera Plataspidae. In 2nd International symposium on Trichogramma and other 1827

egg parasites, 1988, Guangzhou, China. INRA, Paris. 1828

Boggs, C. L., and L. E. Gilbert. 1979. Male contribution to egg production in butterflies: 1829

evidence for transfer of nutrients at mating. Sci. 206: 83-84. 1830

Borah, B.K., and S. K. Dutta. 2002. Entomogenous fungus, Beauveria bassiana (Balsamo) 1831

Vuillemin: a natural biocontrol agent against Megacopta cribrarium (Fab.). Ins. Environ. 1832

8: 7–8. 1833

Borah, B.K., and K. K. Sarma. 2009. Seasonal incidence of negro bug, Megacopta cribrarium 1834

(Fab.) on pigeon pea. Ins. Enviro. 14: 147–149. 1835

Brönmark, C., T. Lakowitz, P. A. Nilsson, J. Ahlgren, C. Lennartsdotter, and J. Hollander. 1836

2012. Costs of inducible defense along a resource gradient. PLOS One: doi: 1837

10.1371/journal.pone.0030467 1838

Ceryngier, P., J. Havelka, and I. Hodek. 2004. Mating and activity of gonads in pre-dormant 1839

and dormant ladybirds (Colecoptera: Coccinellidae). Invert. Repro. Develp. 45: 127-135. 1840

Chatterjee, N. C. 1934. Entomological investigations on the spike disease of sandal (24) 1841

Pentatomidae (Hemipt.) Indian Forest Res. 20: 1- 31. 1842

Chen, G., G. Zhu, and C. Tong. 1996. Soybean yield reduction by Megacopta cribraria and 1843

the threshold index for control. Entomol. Know. 33: 207-208. 1844

Chen, Q., J. Wang, S.-G. Songjing, B. Hongxia, and X. Zuo. 2009. Biological characteristics 1845

of kudzu bug. Henan Agric. Sci. 4: 88-90. 1846

D. Reisig, D. R. Suiter, J. S. Bacheler, K. Kidd, C. H. Ray, X. P. Hu, R. C. Kemerait, E. A. 1847

Scocco, J. E. Eger, J. R. Ruberson, E. J. Sikora, D. A. Herber, Jr., C. Campana, S. 1848

Halbert, S. S. Stewart, G. D. Buntin, M. D. Toews, and C. T. Bargeron. 2013. 1849

Confirmed distribution and occurrence of Megacopta cribraria (F.) (Hemiptera: 1850

Heteroptera: Plataspidae) in the Southeastern United States. J. Entomol. Sci. 48: 118-127. 1851

Davidová-Vilímová, J., and P. Štys. 1980. Taxonomy and phylogeny of West-Palaearctic 1852

Plataspidae (Heteroptera). Studie ČSAV 4: 1-156. 1853

Davidová-Vilímová, J. 2006. Plataspidae. p. 150-165. In: B. Aukema and C. Rieger, [Eds.]. 1854

Catalogue of the Heteroptera of the Palaearctic region. Volume 5: Pentatomomorpha II. 1855

Netherlands Entomological Society, Wageningen. xiii + 550 pp. 1856

1857

Del Pozo-Valdivia, A. I., and D. D. Reisig. 2013. First-generation Megacopta cribraria 1858

(Hemiptera: Plataspidae) can develop on soybeans. J. Econ. Entomol. 3: 533-535. 1859

Denlinger, D. L. 1985. Hormonal control of diapause. In G. A. Kerkut and L. Gilbert (eds.) 1860

Comprehensive Insect Physiology, Biochemistry and PharacologyPergamon. Oxford. 1861

Distant, W.L. 1902. The Fauna of British India, Including Ceylon and Burma. Heteroptera. 1862

Volume 1. Taylor and Francis. London. xxxviii + 438 p. 1863

Easton, E.R., and W.-W. Pun. 1997. Observations on some Hemiptera/Heteroptera of Macau, 1864

South-east Asia. Proc. Entomol. Soc. Washington. 99: 574-582. 1865

Eger, J. E., Jr., L. M. Ames, D. R. Suiter, T. M. Jenkins, D. A. Rider, and S. E. Halbert. 1866

2010. Occurrence of the Old World bug Megacopta cribraria (Fabricius) (Heteroptera: 1867

Plataspidae) in Georgia: a serious home invader and potential legume pest. Insecta 1868

Mundi. 121: 1-11. 1869

Enloe, S. F., and N. J. Loewstein. 2014. Kudzu Control in Residential Areas. ANR-2168, 1870

Alabama Cooperative Extension Service, Auburn, AL. 1871

Esaki, T. 1926. Verzeichniss der Hemiptera-Heteroptera der insel Formosa. Ann. Hist.-Nat. 1872

Musei Nationalis Hungarici 24: 138-189. 1873

Esaki, T., and T. Ishihara. 1951. Hemiptera of Shansi, North China II. Pentatomoidea. Mushi 1874

22: 29-44. 1875

Froeschner, R. C. 1984. Does the Old World family Plataspidae (Hemiptera) occur in North 1876

America. Entomol. News. 95: 36. 1877

Fukatsu, T., and T. Hosokawa. 2002. Capsule-transmitted gut symbiotic bacterium of the 1878

Japanese common plataspid stinkbug, Megacopta punctatissima. App. Enviro. Microbiol. 1879

68: 389–396. 1880

Fukatsu, T., and T. Hosokawa. 2008. Capsule-transmitted obligate gut bacterium of plataspid 1881

stinkbug: a novel model system for insect symbiosis studies pp. 95-113. In K. Bourtzis 1882

and T. A. Miller (eds.), Insect Symbiosis, vol. 3. CRC Press, Taylor & Francis Group, 1883

Boca Raton, FL. 1884

Funayama, K. 2004. Importance of apple fruits as food for the brown-marmorated stink bug, 1885

Halyomorpha halys (Stål) (Heteroptera: Pentatomoidae). Appl. Etomol. Zool. 39: 617- 1886

623. 1887

Gardner, W.A., H. B. Peeler, J. LaForest, P. M. Roberts, A. N. Sparks, Jr., J. K. Greene, D. 1888

Reisig, D. R. Suiter, J. S. Bacheler, K. Kidd, C.H. Ray, X. P. Hu, R. C. Kemerait, E. 1889

A. Scocco, J.E. Eger, J. R. Ruberson, E. J. Sikora, D. A. Herber, Jr., C. Campana, S. 1890

Halbert, S. S. Stewart, G. D. Buntin, M. D. Toews, and C. T. Bargeron. 2013. 1891

Confirmed distribution and occurrence of Megacopta cribraria (F.) (Hemiptera: 1892

Heteroptera: Plataspidae) in the Southeastern United States. J. Entomol. Sci. 48: 118-127. 1893

Gerding, P.M. and E. A. Figueroa. 1989. Hyalomyodes triangulifera Loew (Diptera: 1894

Tachinidae), parasite de Bruchus pisorum L. Agric. Tecnia. (Santiago). 49: 69-70. 1895

Golec, J. R., X. P. Hu, C. H. Ray, and N. E. Woodley. 2013. Strongygaster triangulifera 1896

(Diptera: Tachinidae) as a parsitoid of adults of the invasive Megacopta cribraria 1897

(Heteroptera: Plataspidae) in Alabama. J. Entomol. Sci. 48: 1-3. 1898

Golec, J. R., X. P. Hu, C. Ray, and N. E. Woodley. 2013. Strongygaster triangulifera (Diptera: 1899

Tachinidae) as a Parasitoid of Adults of the Invasive Megacopta cribraria (Heteroptera: 1900

Plataspidae) in Alabama. J. Entomol. Sci. 48: 1-3. 1901

Golec, J., X. P. Hu, and T. Reed. 2014a. Kudzu bug control in residential areas: frequently 1902

asked questions (FAQ). Entomology Series Timely Information, Agricultural & Natural 1903

Resources, ANR-2163. Alabama Cooperative Extension Service, Auburn, AL. 1904

Golec, J., T. Reed, L. Yang, and X. P. Hu. 2014b. Kudzu bug control in soybeans: frequently 1905

asked questions (FAQ). Entomology Series Timely Information, Agricultural & Natural 1906

Resources, ANR-2176. Alabama Cooperative Extension Service, Auburn AL. 1907

Greene, J. K., P. M. Roberts, W. A. Gardner, F. Reay-Jones, and N. Seiter. 2012. Kudzu 1908

bug identification and control in soybeans. United Soybean Board, Chesterfield, MO. 1909

Online http://digital.turn-page.com/i/87846 1910

Greenestone, M. H., P. G. Tillman, and J. S. Hu. 2013. Predation of the newly invasive pest 1911

Megacopta cribraria (Hemiptera: Plataspidae) in soybean habitats adjacent to cotton by a 1912

complex of predators. J. Econ. Entomol. 107: 947-954. 1913

Grenier, S., and G. Plantevin. 1990. Development modifications of the parasitoid 1914

Pseudoperichaeta nigrolineata (Diptera: Tachinidae) by fenoxycard, an insect growth 1915

regulator, applied onto its host Ostrinia nubilalis (Lep., Pyralidae). J. App. Entomol. 1916

110: 462-470. 1917

Grenier, S., and G, Plantevin. 1991. Action of an insect growth regulator, fenoxycard, on the 1918

parasitoid Pseudoperichaeta nigrolineata (Diptera: Tachinidae). Redia. 74: 425-431. 1919

Guimarães, J.H. 1978. Hyalomyodes brasiliensis Townsend (Diptera: Tachinidae), a parasite of 1920

the introduced pest Lagria villosa Fabricius (Coleoptera: Coccinellidae) in western North 1921

America. Can. Entomol. 130: 905-906. 1922

Härding, R., T. Gosden, and R. Aguilée. 2008. Male mating constraints affect mutual mate 1923

choice: prudent male courting and sperm-limited females. Am. Nat. 172: 259-271. 1924

Hasegawa, H. 1965. Major pests of economic plants in Japan. Jpn. Plant Prot. Assoc. Tokyo. 1925

412 p. 1926

Henry, T. J. 2009. Biodiversity of the Heteroptera. In Insect biodiversity, 1st edition: R. G. 1927

Footit and P. H. Alder (eds.) Science and Society. Wiley-Blackwell Publishing. 223-263 1928

pp. 1929

Hibino, Y. 1986. Female choice for male gregariousness in a stink bug, Megacopta 1930

punctissimum [sic!] (Montandon) (Heteroptera: Plataspidae). J. Ethol. 3: 91-95. 1931

Hibino, Y., and Y. Ito. 1983. Mating aggregation of a stink bug, Megacopta punctissimum 1932

(Montandon) (Heteroptera: Plataspidae). Res. Popul. Ecolo. 25: 180-188. 1933

Hirashima, Y. 1989. A check list of Japanese insects. Vol I. Faculty of Agriculture, Kyushu 1934

University & Center for the Study of Japanese Field Life,n Fukuoka, 540 p. 1935

Hoffmann, W. E. 1931. Notes on Hemiptera and Homoptera at Canton, Kwangtung Province, 1936

Southern China 1924-1929. USDA Insect Pest Survey Bulletin 11: 138- 151. 1937

Hoffmann, W. E. 1932. Notes on the binomics of some Oriental Pentatomidae (Hemiptera) 1938

Arch. Zool. Ital. 16: 1010-1027. 1939

Hoffmann, W. E. 1935. An abridged catalogue of certain Scutelleroidea (Plataspidae, 1940

Scutelleridae, and Pentatomidae) of China, Chosen, Indo-China, and Taiwan. Linnan Uni. 1941

Sci. Bul. 7: 1-294. 1942

Horn S., and J. L. Hanula. 2011. Influence of trap color on Collection of the recently- 1943

introduced Bean Plataspid, Megacopta cribraria (Hemiptera: Plataspidae). J. Entomol. 1944

Sci. 46: 85-87. 1945

Horton, D. 2013. Georgia Pest Management Handbook. Commercial Ed., special bulletin 28. 1946

Published by the Uni. of GA. Cooperative Extension. 1947

Hosokawa, T., and N. Suzuki. 1999. Mating aggregation and copulatory success by males of 1948

the stink bug, Megacopa punctatissima (Heteroptera: Plataspidae) Jpn. Soc. Appl. 1949

Entomol. Zool. 35: 93-99. 1950

Hosokawa, T., and N. Suzuki. 2001. Significance of Prolonged Copulation Under the 1951

Restriction of Daily Reproductive Time in the Stink Bug Megacopta punctatissima 1952

(Heteroptera: Plataspidae). Ann. Entomol. Soc. Am. 94: 750-754. 1953

Hosokawa, T., Y. Kikunchi, N. Nikoh, M. Shimada, and T. Fukatsu. 2006. Strict host- 1954

symbiot cospeciation and reductive genome evolution in insect gut bacteria. PLOS Biol. 1955

4: 1841-1851. 1956

Hosokawa, T., Y. Kikunchi, M. Shimada, and T. Fukatsu. 2007. Obligate symbiont involved 1957

in pest status of host insect. Proc. R. Soc. Biol. Sci. (B) 274: 1979-1984. 1958

Hosokawa, T., N. Nikoh, and T. Fukatsu. 2014. Fine-scale geographical origin of an insect 1959

pest invading North America. PLOS ONE doi:0.1371/journal.pone.0089107. 1960

Hsiao, T.-Y., and S.-Z. Ren. 1977. A handbook for the determination of the Chinese Hemiptera- 1961

Heteroptera. Volume 1. Scientific Publishing Co., Beijing. 330 p. 1962

Hu, X. P., and D. Carroll. 2012. Alabama soybean: kudzu bug life cycle diversified in terms of 1963

hosts. http://agfax.com/2012/05/18/Alabama-soybean-kudzu-bugs-making-their-move/ 1964

Hu, X.P. 2014. Kudzu bugs are on moving, with a little dent on population made by the chilly 1965

winter. Online: https://sites.aces.edu/group/timelyinfo/Documents/TI-4th-ColdImpact- 1966

20140319.pdf 1967

Ishihara, T. 1937. A list of Heteroptera from Hiroshima Prefecture. Part I. The Entomol. Wrld. 1968

5: 475-492. 1969

Ishihara, T. 1950. The developmental stages of some bugs injurious to the kidney bean 1970

(Hemiptera) Trans. Shikoku Entomol. Soc. 1: 17-31. 1971

Jenkins, T., D. Suiter, J. Eger, L. Ames, D. Buntin, and T. Eaton. 2010. The preliminary 1972

genetics of an invasive true bug from the Old World: Implication for the New World. J. 1973

Entomol. Sci. 45: 1-2. 1974

Jenkins, T. M., and T. D. Eaton. 2011. Population genetic baseline of the first plataspid stink 1975

bug symbiosis (Hemiptera: Heteroptera: Plataspidae) reported in North America. Insects. 1976

2: 264-272. 1977

Johnson, N.F. 1996. Revision on world species of Paratelenomus Dodd (Hymenoptera: 1978

Scelionidae). Can. Entomol. 128: 272-291. 1979

Jones, W. A., Jr., and M. J. Sullivan. 1981. Overwintering habitats, spring emergence 1980

patterns and winter mortality of some South Carolina Hemiptera. Environ. Entomol. 11: 1981

867-875. 1982

Kehat, M., and M. Wyndham. 1972. The effect of food and water on development, longevity, 1983

and fecudintiy in the Rutherglen bug, Nysius vinitor (Hemiptear: Lygaeidae). Austr. J. 1984

Zool. 20: 119:130. 1985

Kester, K. M., and C. M. Smith. 1984. Effect of diet on growth, fecundity and duration of 1986

tethered flight of Nezara viridula. Entomol. Exp. Appl. 35: 75-81. 1987

Kevan, D. K. McE. and R. T. B. Koshnaw. 1988. Hyalomyodes (Diptera: Tachinidae), an 1988

endoparasite of Tetrigoidea (Orthoptera). Entomol. Rec. 100: 55-57. 1989

Kikuchi, A., and H. Kobayashi. 2010. Effect of injury by adult Megacopta punctatissima 1990

(Montandon) (Hemiptera: Plataspidae) on the growth of soybean during the vegetative 1991

stage of growth (English abstr.). Jpn. J. Appl. Entomol. Zool. 54: 37–43. 1992

Kikuchi, Y., T. Hosokawa, and T. Fukatsu. 2008. Diversity of bacterial symbiosis in 1993

stinkbugs. In T. V. Dijk (ed.) Microbial Ecology Research Trends. Nova Science 1994

Publishers Inc., N. Y., pp. 39-63. 1995

Kim, H. R., and C. E. Lee. 1993. The spermathecae of the Plataspidae from Korea. Nat. Life 1996

(Korea). 23: 115-120. 1997

Kiritani, K., N. Hokyo, and K. Kimura. 1966. Factors affecting the winter mortality in the 1998

southern green stink bug, Nezara viridula L. Ann. Soc. Entomol. Frn. 2: 199–207. 1999

Kirkaldy, G. W. 1910. A list of the Hemiptera of oriental China. Part II. Ann. Soc. Entomol. 2000

Bel. 54: 103-112. 2001

Kitamura, C., S. Wakamura, and S. Takahashi. 1984. Identification and functions of ventral 2002

glands secretion of some Heteroptera. Appl. Entomol. Zool. 19: 33-41. 2003

Kment, P., and J. Vilímová. 2010. Thoracic scent efferent system of Pentatomoidea 2004

(Hemiperta: Heteroptera): a review of terminology. Zootaxa: 1-77. 2005

Kobayashi, T. 1981. Insect pests of soybeans in Japan. Miscel. Public. Hohoku Natl. Agric. 2006

Experimental Stations. 2: 1-39. 2007

Kobayashi, T., and M. H. Osakabe. 2008. Pre-winter copulation enhances overwintering 2008

success of Orius females (Heteroptera: Anthocoridae). Appl. Entomol. Zool. 44: 47-52. 2009

Kono, S. 1990. Spatial distribution of three species of sting bugs attacking soybean seeds. Jpn. J. 2010

Appl. Entomol. Zool. 34: 89-96. 2011

Konstantinou, I. K., A. K. Zarkadis, and T. A. Albanis. 2001. Photodegradation of selected 2012

herbicides in various natural waters and soils under environmental conditions. J. Environ. 2013

Qual. 30: 121-130. 2014

Koshiyama, Y., H. Tsumuki, M. Muraji, K. Fujisaki, and F. Nakasuji. 1993. Transfer of 2015

male secretion to females through copulation in Mendia scotti (Heteroptera, 2016

Pentatomidae). Appl. Entomol. Zool. 28: 325-332. 2017

Koshiyama, Y., K. Fujisaki, and F. Nakasuji. 1994. Mating and diapause in hibernating 2018

adults of Mendia scotti Puton (Heteroptera: Pentatomidae). Res. Popul. Ecol. 36: 87-92. 2019

Koshiyama, Y., K. Fujisaki, and F. Nakasuji. 1996. Nutritional contribution of females of 2020

14C-labed male secretions transferred during mating in Mendia scottyi (Heteroptera, 2021

Pentatomidae). Res. Popul. Ecol. 38: 51-56. 2022

Koshiyama, Y., K. Fujisaki, and F. Nakasuji. 1997a. Effect of mating during hibernation on 2023

life history traits of female adults of Mendia scotti (Heteroptera, Pentatomidae). Chugoku 2024

Kontyu 11: 11-18 (Japanese with English summary). 2025

Koshiyama, Y., K. Fujisaki, and F. Nakasuji. 1997b. Benefits of mating during hibernation 2026

for male adults of Mendia scotti (Heteroptera, Pentatomidae) Chugoku Kontyu 11: 1-9 2027

(Japanese with English summary). 2028

Kott, P., S. Roth, and K. Reinhardt. 2000. Hibernation mortality and sperm survival during 2029

dormancy in female Nabidae (Heteroptera: Nabidae). Opus. Zool. Fluminen. 182: 1-6. 2030

Lal, O.P. 1980. A compendium of insect pests of vegetables in India. Bull. Entomol. Soc. India 2031

16: 52-88 2032

Leong, K. H. L., M. A. Yoshimura, and C. Williams. 2012. Adaptive significance of 2033

previously mated monarch butterfly females (Danaus plesippus (L.)) overwintering at a 2034

California winter site. J. Lep. Soc. 4: 205-210. 2035

Li, Y. H., Z. S. Pan, J. P. Zhang, and W. S. Li. 2001. Observation of biology and behavior of 2036

Megacopta cribraria (Fabricius). Plant Prot. Technol. Ext. 21: 11-12. 2037

Matsumura, S. 1910. Die schädlichen und nützlichen Insekten vom Zuckerrohr Formosasa. 2038

Zeitchrift für Wiseenschaftliche Insektbiologie 6: 136-139. 2039

Mattson, W. J., Jr. 1990. Herbivory in relation to plant nitrogen content. Annu. Rev. Ecol. Syst. 2040

11: 119-61. 2041

McPherson, R. M. J. R. Pitis, L. D. Newson, J. B. Chapin, and D. C. Herzog. 1982. 2042

Incidence of Tachinid parasitism of several stink Bug (Heteroptera: Pentatomidae) 2043

species associated with soybean. J. Econ. Entomol. 75: 783-786. 2044

Medal, J., S. Halbert, T. Smith and A. S. Cruz. 2013. Suitability of selected plants to the Bean 2045

Plataspid, Megacopta cribraria (Hemiperta: Plataspidae) in no-choice tests. Fl. Entomol. 2046

Soc. 96: 631-633. 2047

Miyamoto, S. 1961. List of Ovariole numbers in Japanese Heteroptera. Sieboldia 2: 69-82. 2048

Moizuddin, M., and I., Ahmad. 1975. Eggs and nymphal systematics of Coptsoma cribrarium 2049

(Fabr.) (Pentatomoidea: Plataspidae) with a note on other plataspids and their phylogeny. 2050

Rec. Zool. Sur. Pak. 7: 93-100. 2051

Moizuddin, M., and I. Ahmad. 1977. Eggs and nymphal systematics of Coptosoma cribrarium 2052

(Fabr.) (Pentatomoidea: Plataspidae) with a note on other plataspids and their phylogeny. 2053

Rec. Zool. Sur. Pak. 7: 93-100. 2054

Montandon, A. L. 1896. Plataspidinae. Nouvelle série d’études et descriptions. Ann. Soc. 2055

Entomol. Belg. 40: 86-134. 2056

Montandon, A. L. 1897. Les Plataspidines du Muséum d’histoire naturelle de Paris. Ann. Soc. 2057

Entomol. Fran. 1896: 436-464. 2058

Müller, H. J. 1956. Experimentelle studien an der symbiose von Coptosoma scutellatum Geoffr. 2059

(Hem Heteropt.) Z. Morphol. Ökol. Tiere. 44: 459-482. 2060

Musser, F. R., A. L. Catchot, Jr., J. A. Davis, D. A. Herbert, Jr., G. M. Lorenz, T. Reed, D. 2061

D. Reisig, and S. D. Stewart. 2012. 2011 Soybean insect losses in the Southern US. 2062

Assoc. Midsouth Entomol., Miss. Entomol. Assoc. 5: 11-22. 2063

Musser, F. R., A. L. Catchot, Jr., J. A. Davis, D. A. Herbert, Jr., G. M. Lorenz, T. Reed, D. 2064

D. Reisig, and S. D. Stewart. 2013. 2012 Soybean insect losses in the Southern US. 2065

Assoc. Midsouth Entomol. Miss. Entomol. Assoc. 6: 12-24. 2066

Panizzi, A. R., M. H. M. Galileo, H. A. O. Gastal, J. F. F. Toledo, and C. A. Wild. 1980. 2067

Dispersal of Nexara viridula and Piezodorus guildinii nymphs in soybeans. Enviro. 2068

Entomol. 9: 293-297. 2069

Panizzi, A. R. and F. Slansky, Jr. 1991. Suitability of selected legumes and the effect of 2070

nymphal and adult nutrition in the southern green stink bug (Hemiptera: Heteroptera: 2071

Pentatomidae). J. Econ. Entomol. 84: 103-113. 2072

Panizzi, A. R., and S. I. Saraiva. 1993. Performance of nymphal and adult southern green stink 2073

bug on an overwintering host plant and impact of nymph to adult food-switch. Entomol. 2074

Exp. Appl. 68: 109-115. 2075

Panizzi, A. R., and E. Hirose. 1995. Survival, reproduction, and starvation resistance of adult 2076

southern green stink bug (Heteroptera: Pentatomidae) reared on sesame or soybean. Ann. 2077

Entomol. Soc. Am. 88: 661-667. 2078

Panizzi, A. R. 1997. Wild hosts of pentatomids: ecological significance and role in their pest 2079

status on crops. Annu. Rev. Entomol. 42: 99-122. 2080

Parker, G. A. 1970. Sperm competition and its evolutionary consequences in the insects. Biol. 2081

Rev. 45: 164-170. 2082

Pendergrast, J. G. 1957. Studies on the reproductive organs of the Heteroptera with a 2083

consideration of their bearing on classification. Trans. R. Entoml. Soc. Lond. 109: 1-63. 2084

Pener, M. P. 1992. Environmental cues, endocrine factors, and reproductive diapause in male 2085

insects. Chron. Int. 9: 102-113. 2086

Pinto, S. B., and A. R. Panizzi. 1994. Performance of nymphal and adult Euscistus heros (F.) 2087

on milkweed and on soybean and effect of food switch on adult survivorship, 2088

reproduction and weight gain. An. Soc. Entomol. Brasil. 23: 549- 555. 2089

Purrington, F.F., D. M. Pavuk, R. P. Herd, and B. R. Stinner. 1990. First scarab host for 2090

Strongygaster triangulifera (Diptera: Tachinidae): the dung beetle, Aphodius fimertarius 2091

(Coleoptera: Scarbaeidae). The Great Lakes Entomol. 23: 171-172. 2092

Rajmohan, A., and T. C. Narendran. 2001. Parasitoid complex of Coptosoma cribrarium 2093

(Fabricius) (Plataspididae: Hemiptera). Ins. Environ. 6: 163. 2094

Ramakrishna Ayyar, T. V. 1913. On the life history of Coptosoma cribraria Fabr. J. Bombay 2095

Nat. Hist. Soc. 22: 412-414. 2096

Reeves, W.K. and J. E. O’hara. 2004. First report of Strongygaster triangulifera 2097

(Diperta: Tachinidae) as a parasitoid of a cantharid beetle, Chauliognathus 2098

pennsylvanicus (Coleoptera: Cantharidae). Can. Entomol. 136: 661-662. 2099

Reisig, D. D., and J. Bacheler. 2012. Kudzu bug (Megacopta cribraria), a new potentially 2100

devastating pest of soybeans. Produced by North Carolina Statue University Extension 2101

Entomology. Available online 2102

http://www.kudzubug.org/docs/NC_Growers_summer_2012.pdf 2103

Rekha, S., and C. P. Mallapur. 2007. Abundance and seasonality of sucking pests of dolichos 2104

bean. Karmataka J. Agric. Sci. 20: 397-398. 2105

Ren, S. 1984. Studies on the fine structure of egg-shells and the biology of Megacopta Hsiao et 2106

Jen from China (Hemiptera: Plataspidae) Entomotaxonomia 6: 327-332. 2107

Roberts, P. 2013. Agent Update, Kudzu Bug, Megacopta cribraria. Produced by University of 2108

Georgia Extension. Available online: http://www.kudzubug.org/docs/GA- 2109

AgentUpdate_5-31-2013.pdf 2110

Roberts, P., and J. Whitaker. 2012. Kudzu bug management. Available online: 2111

http://www.kudzubug.org/docs/GA_1-2012_SBGrow.pdf 2112

Roth, S., and K. Reinhardt. 2003. Facultative sperm storage in response to nutritional status in 2113

a female insect. Proc. R. Soc. Lond. B. (Suppl.) 270: S54-S56. 2114

Ruano, F., C. Mercedes, A. J. Sánchez-Raya, and A. Peña. 2010. Olive trees protected from 2115

the olive bark beetles, Phloeotribus scarabaeoides (Bernard 1788) (Coleoptera, 2116

Curculionidae, Scolytinae) with a pyrethroid insecticide: Effects of the insect community 2117

of the olive grove. Chemosphere. 80: 35-40. 2118

Ruberson, J. R., K. Takasu, G. D. Buntin, J. E. Eger Jr., W. A. Gardner, J. K. Greene, T. 2119

M. Jenkins, W. A. Jones, D. M. Olson, P. M. Roberts, D. R. Suiter, and M. D. 2120

Toews. 2012. From Asian curiosity to eruptive American pest: Megacopta cribraria 2121

(Hemiptera: Plataspidae) and prospects for its biological control. App. Entomol. Zool. 48: 2122

3-13. 2123

Saulich, A. Kh., and D. L. Musolin. 2011. Diapause in the seasonal cycle of stink bugs 2124

(Heteroptera, Pentatomidae) from the temperate zone. Entomol. Rev. 92: 1-16. 2125

Schaefer, C. W., and A. R. Panizzi (eds.). 2000. Heteroptera of Economic Importance. CRC 2126

Press LLC, New York, NY. Press 856 pp. 2127

Schuh, R. T., and J. A. Slater. 1995. True Bugs of the World (Hemiptera: Heteroptera). 2128

Classification and Natural History. Cornell University Press, Ithaca, New York, U.S.A 2129

336 pp. 2130

Schumann, F. W., and J. W. Todd. 1982. Population dynamics of the southern green stink bug 2131

(Heteroptera: Pentatomidae) in relation to soybean phenology. J. Econ. Entomol. 75: 748- 2132

753. 2133

Scriber, J. M., and F. Slansky, Jr. 1981. The nutritional ecology of immature insects. Ann. 2134

Rev. Entomol. 26: 183- 211. 2135

Seiter, N. J., E. P. Benson, F. P. F. Reay-Jones, J. K. Greene, and P. A. Zungoli. 2013a. 2136

Residual efficacy of insecticides applied to exterior building material surfaces for control 2137

of nuisance infestations of Megacopta cribraria (Hemiptera: Plataspidae). J. Econ. 2138

Entomol. 106: 2448-2456. 2139

Seiter, N. J., F.P.F. Reay-Jones, and J. K. Greene. 2013b. Within-field spatial distribution of 2140

Megacopta cribraria (Hemiptera: Plataspidae) in soybean (Falbes: Fabaceae). Environ. 2141

Entomol. 42: 1363-1374. 2142

Smith, R. 2013. Kudzu bug management and control in Alabama soybean. 2143

http://www.aces.edu/anr/crops/weed-pest/documents/KudzuBugbyRonSmith.pdf 2144

Steiner, U. K., and T. Pfeiffer. 2007. Optimizing time and resource allocation trade-offs for 2145

investment into morphological and behavioral defense. Am. Nat. 118-129 2146

Suiter, D. R., J. E. Eger, Jr., W. A. Gardner, R. C. Kemerait, J. N. All, P. M. Roberts, J. K. 2147

Greene, L. M. Ames, G. D. Buntin, T. M. Jenkins, and G. K Douce. 2010. Discovery 2148

and distribution of Megacopta cribraria (Hemiptera: Heteroptera: Plataspidae) in 2149

northeast Georgia. J. Integ. Pest Mngmt. doi: 10.1603/ipm10009 2150

Shapiro, A. M. 1982. Survival of refrigerated Tatochila butterflies (Lepidoptera: Pieridae) as 2151

an indicator of male nutrient investment in reproduction. Oecologia 53: 139-140. 2152

Shima, H. 1999. Host-parasite catalog of Japanese Tachinidae (Diptera). Makunagi/ Acta 2153

Dipterol. Sup. 1: 1-108. 2154

Shroff, K. D. 1920. A list of the pests of pulses in Burma. p. 343-346. In: T. B. Fletcher (ed.) 2155

Proc. 3rd Entomol. Meet., Pusa. 1919 Vol. 1. 417 p. 2156

Simmons, L. W., and M. T. Siva-Jothy. 1998. Sperm competition in insects: mechanisms and 2157

the potential for selection. In: Birkhead, T., and Møller, A. (eds.) Sexual selection and 2158

sperm competition. San Diego: Academic Press; 503–564. 2159

Soares, M. A., J. D. Batista, J. C. Sanuncio, J. Lino-Neto, and J. E. Serrão. 2011. Ovary 2160

development, egg production and oviposition for mated and virgin females of the 2161

predator Podisus nigrispinus (Heteroptera: Pentatomidae). Act. Sci. Agric. 33: 597-602. 2162

Socha, R. 2010. Pre-diapause mating and overwintering of fertilized adult females: new aspects 2163

of the life cycle of the wing-polymorphic bug, Pyrrhocoris apterus (Heteroptera: 2164

Pyrrhocoridae). Eur. J. Entomol. 107: 521-525. 2165

Suiter, D. R., J. E. Eger, Jr., W. A. Gardner, R. C. Kemerait, J. N. All, P. M. Roberts, J. K. 2166

Greene, L. M. Ames, G. D. Buntin, T. M. Jenkins, and G. K. Douce. 2010. Discovery 2167

and distribution of Megacopta cribraria (Hemiptera: Plataspidae) in Northeast Georgia. 2168

J. Integ. P. Manag. 1: DOI: 10.1603/IPM10009. 2169

Suiter, D. R., L. M. Ames, J. E. Eger, Jr., and W. A. Gardner. 2010b. Megacopta cribraria 2170

as a nuisance pest. UGA-CAES Extension Circular No. 991, University of Georgia, 2171

Athens, GA. 2172

Sujithra, M., S. Srinivasan, and K. V. Hariprasad. 2008. Outbreak of lablab bug, Coptosoma 2173

cribraria Fab. on field bean, Lablab purpureus var. lignosus Medikus. Ins. Environ. 14: 2174

77-78. 2175

Takagi, M., and K. Murakami. 1997. Effect of temperature on development of Paratelenomus 2176

saccharalis (Hymenoptera: Scelionidae), an Egg Parasitod of Megacopta punctatissimum 2177

(Hemiptera: Plataspidae). Appl. Entomol. Zool. 32: 659-660. 2178

Takasu, K., and Y. Hirose. 1985. Seasonal egg parasitism of phytophagous stink bugs in a 2179

soybean field in Fukuoka (In Japanese with English summary). Proc. Assoc. Plant Prot. 2180

Kyushu, 31: 127–131. 2181

Tauber, M. J., C. A. Tauber, and S. Masaki. 1986. Seasonal Adaptations of Insects. Oxford 2182

University Press, New York. 411 pp. 2183

Taylor, F. 1984. Mexican bean beetles mate successfully in diapause. Intl. J. Invert. Repro. 7: 2184

297-302. 2185

Tayutivutikul, J., and K. Yano. 1990. Biology of insects associated with the kudzu plant, 2186

Pueraria lobata (Leguminosae). 2. Megacopa punctissimum [sic!] (Hemiptera, 2187

Plataspidae). Jpn. J. Entomol. 58: 533-539. 2188

Tayutivutikul, J., and K. Kusigemati. 1992. Biological studies of insects feeding on the kudzu 2189

plant, Pueraria lobata (Leguminosae) I. list of feeding species. Memoirs of the Faculty of 2190

Agriculture, Kagoshima University 28: 89-124. 2191

Tayutivutikul, J., and K. Kusigemati. 1992. Biological studies of insects feeing on the kudzu 2192

plant, Pueraria lobata (Leguminosae). II. seasonal abundance, habitat development. 2193

South Pacific Study 12: 37-88. 2194

Tayutivutikul, J., and K. Yano. 1990. Biology of insects associated with the kudzu plant, 2195

Pueraria lobata (Leguminosae). 2. Megacopa punctissimum [sic!] (Hemiptera, 2196

Plataspidae). Jpn. J. Entomol. 58: 533-539. 2197

Thejaswi, L., M. I. Naik, and M. Manjunatha. 2008. Studies on population dynamics of pest 2198

complex of field bean (Lablab purpureus L.) and natural enemies of pod borers. 2199

Karnataka J. Agric. Sci. 21: 339-402. 2200

Thippeswamy, C., and B. K. Rajagopal. 1998. Assessment of losses caused by the lablab bug, 2201

Coptosoma cribraria (Fabricius) to the field bean Lablab purpureus var. lignosus 2202

Medikus. Karnataka J. Agric. Sci. 11: 941-946. 2203

Thippeswamy, C., and B. K. Rajagopal. 2005a. Life history of the lablab bug, Coptosoma 2204

cribraria on the field bean Lablab purpureus var. lignosus Medikus. Karnataka J. Agric. 2205

Sci. 18: 39-43. 2206

Thippeswamy, C., and B. K. Rajagopal. 2005b. Comparative biology of Coptosoma cribraria 2207

Fabricius on field bean, soy bean and redgram. Karnataka J. of Ag. Sci. 18: 138-140. 2208

Thompson, W. R. 1954. Hyalomyodes triangulifera Loew (Diptera: Tachinidae). Can. Entomol. 2209

86: 137-144. 2210

Tollrian R., and C. D. Harvell. 1999. The ecology and evolution of inducible defenses. 2211

Princeton University Press. 2212

U. S. Environmental Protection Agency (EPA). 2013. Environmental hazard and general 2213

labeling for pyrethroid and synergized pyrethrin non-agricultural outdoor products. 2214

http://www.epa.gov/oppsrrd1/reevaluations/enviromental-hazard-statements.html 2215

Velasco, L. R. I., and G. H. Watler. 1993. Potential of host-switching in Nezara viridula 2216

(Hemiptera: Pentatiomidae) to enhance survival and reproduction. Environ. Entomol. 22: 2217

326-333. 2218

Villas Bôas, G. L., and A. R. Panizzi. 1980. Biologia de Euschistus heros (Fabricius, 1798) em 2219

soja (Glycine max (L.) Meriill). An. Soc. Entomol. Brasil. 9: 105 113. 2220

Vilímová, J., and K. Kutalova. 2011. Occurrence of certain cuticular structures confirms 2221

functionality of dorsal abdominal scent glands in Acanthosomatidae (Heteroptera: 2222

Pentatomoidea) Bul. Entomol. Res. 102: 29-42. 2223

Voegelé, J. 1969. Les Aelia du Maroc. Al. Awamia. 30: 1-136. 2224

Waldvogel, M., and P. Alder. 2012. Kudzu bug – A nuisance and agricultural pest. residential, 2225

structural and community pests. North Carolina Dept. of Entomology. Insect Notes. 2226

Online: http://www.ces.ncsu.edu/depts/ent/notes/Urban/kudzubug.htm 2227

Wall, R.E. 1928. A comparative study of a chalcid egg parasite in three species of Plataspidinae. 2228

Lingnan Sci. J. 6: 231–239. 2229

Wall, R.E. 1931. Dissolcus tetartus Crawford, a scelionid egg parasite of Plataspidinae in China. 2230

Lingnan S. J. 9: 381–382. 2231

Wang, H. S., C. S. Zhang, and D. P. Yu 2004. Preliminary studies on occurrence and control 2232

technology of Megacopta cribraria (Fabricius). China Plant. Prot. 22: 7-9. 2233

Wang, Z.-X., H.-D. Wang, G.-H. Chen, Z.-G. Zi, and C.-W. Tong. 1996. Occurrence and 2234

control of Megacopta cribraria (Fabricius) on soybean. Plant Prot. 22: 7-9. 2235

Wong, A. C. M., and S. T. Mak. 2012. Preseptal cellulitis in a child caused by Megacopta 2236

centrosignatum. J. Am. Assoc. Ped. Ophthalmol. Strab. 16: 577-578. 2237

Wu, M.-X. and K.-T. Xu. 2002. Preliminary studies on the pests of green soybean in Fuzhou 2238

suburbs. Wuyi Sci. J. 18: 28:33. 2239

Wu, M.-X., and K.-T. Xu. 2002. Preliminary studies on the pests of green soybean in Fuzhou 2240

suburbs. Wuyi Sci. J. 18: 28-33. 2241

Wu, M.-X., Z.-Q. Wu, and S.-M. Hua. 2006. A preliminary study on some biological 2242

characters of the globular stink bug, Megacopta cribraria, and its two egg parasitoids. J. 2243

Fujian Agric. Forest. Uni. Nat. Sci. ed. 35: 147-150. 2244

Xing, G.-N., B. Zhou, T.-J. Zhao, D.-Y. Yu, GH. Xing, S.-Y. Chen, and J.-Y. Gai. 2008. 2245

Mapping QTLs of resistance to Megactopa cribraria (Fabricius) in soybean. Acta. Agric. 2246

Sin. 34: 361-368. 2247

Xing, G.-N., T.-J. Zhao, and J.-Y. Gai. 2006. A preliminary study on some biological 2248

character of globular sting bug, Megacopta cribraria and its two egg parasitoids. J. 2249

Fujian Agric. Forest. Uni. Nat. Sci. ed. 35: 147-150. 2250

Yang, W-I. 1934. Revision of Chinese Plataspidae. Bul. Fan Memor. Inst. Biol. 5: 137–236. 2251

Zhang, C.-S., and D.-P. Yu. 2005. Occurrence and control of Megacopta cribraria (Fabricius). 2252

China Country’s Well off Technology 1: 35. 2253

Zhang, Y.-T., X.-G. Du, M. Dong, and W. Shao. 2003. A preliminary investigation of egg 2254

parasitoids of Megactopa cribraria in soybean fields. Entomol. Knowl. 40: 443-445. 2255

Zhang, Y., J. L. Hanula, and S. Horn. 2012. The biology and preliminary host rage of 2256

Megacopta cribraria (Heteroptera: Plataspidae) and its impact on kudzu growth. Environ. 2257

Entomol. 41: 40-50. 2258

Zhu, D.-H., S.-S. Cui, Y.-S. Fan, and Z. Liu. 2013. Adaptive strategies of overwintering adults: 2259

Reproductive diapause and mating behavior in a grasshopper, Stenocatantops splendens 2260

(Othoptera: Cantanopidae). J. Ins. Sci. 23: 235-244. 2261

Xing, G. N., B. Zhou, T. J. Zhao, D. Y. Yu, G. H. Xing, S. Y. Chen, and J. Y. Gai. 2008. 2262

Mapping QTLs of resistance to Megactopa cribraria (Fabricius) in soybean. Acta. Agric. 2263

Sin. 34: 361-368. 2264

2265