Southeastern Naturalist 57 S.M. Huskisson, K.L. Fogg, T.L. Upole, and C.B. Zehnder 22001155 SOUTHEASTERN NATURALIST Vo1l4.( 114):,5 N7–o6. 51 Seasonal Dynamics and Plant Preferences of Megacopta cribraria, an Exotic Invasive Insect Species in the Southeast Sarah M. Huskisson1, Kayla L. Fogg1, Taylor L. Upole1, and Caralyn B. Zehnder1,* Abstract: - Megacopta cribraria (Kudzu Bug) (Hemiptera: Plataspidae) is an invasive insect in the Southeast. In Baldwin County, GA, we found adult Kudzu Bugs at high abundances throughout the summer, while juveniles showed peaks in early July and September. We conducted a series of choice-preference tests and determined that the insects preferred Glycine max (Soybean) over Phaseolus lunatus (Lima Bean). Additionally, they preferred Pueraria montana var. lobata (Kudzu) over Soybeans, highlighting the role of Kudzu as a primary host plant. Additionally, Kudzu Bugs continued to feed on Soybeans, even in the presence of Kudzu. Introduction Megacopta cribraria (F.) (Hemiptera: Plataspidae) (Kudzu Bug) is an invasive insect species in Georgia and throughout the southeastern US. The Kudzu Bug was first documented in 2009 in 9 northeast Georgia counties (Suiter et al. 2010). In the fall of 2010, Kudzu Bugs were confirmed in more than 60 Georgia counties in addition to North Carolina and South Carolina (Suiter et al. 2010), and they have subsequently spread into 8 southeastern states, including South Carolina, Georgia, North Carolina, Alabama, Virginia, Tennessee, Florida, and Mississippi (Gardner et al. 2013). The most frequent host plant of the Kudzu Bug is Pueraria montana var. lobata (Willd.) Maesen & S. Almeida (Kudzu), though it commonly feeds on other legume species, including economically important crops like Glycine max (L.) Merrill (Soybean). The Kudzu Bug is native to Asia, and, even though it is widely distributed there, it is not usually a major pest of legumes in its native home range (Ruberson et al. 2013). In the southeastern US, it thrives on the abundant Kudzu, which was introduced into the US from Japan in 1876 and has since spread across the region (Forseth and Innis 2004). The Kudzu Bug is the only member of its family (Plataspidae) in the Western Hemisphere (Ruberson et al. 2013). Megacopta cribraria is known by many common names, including Kudzu Bug, Bean Plataspid, Globular Stinkbug, and Lablab Bug, because it feeds on the legume Lalab purpureus (L.) Sweet (Lablab) in its native range. The insect is grayish-brown in color and has a square “shield” on its back; adults are 4–6 mm long (Eger et al. 2010). Kudzu Bugs release an offensive odor when disturbed, and they produce a substance when crushed that can stain cloth and wood. Additionally, the nymphs can cause minor skin irritation (Ruberson et al. 2013). Kudzu Bugs are attracted to light-colored surfaces, 1Department of Biological and Environmental Sciences, CBX 081, Georgia College and State University, Milledgeville, GA 30161. *Corresponding author - caralyn.zehnder@gcsu.edu. Manuscript Editor: Robert Jetton Southeastern Naturalist S.M. Huskisson, K.L. Fogg, T.L. Upole, and C.B. Zehnder 2015 Vol. 14, No. 1 58 including houses, where they often congregate in high densities, attracting the attention of homeowners and contributing to their classification as an urban pest species (Ruberson et al. 2013). The life history of the Kudzu Bug has been very well described by Eger et al. (2010). Kudzu Bugs overwinter as adults clustered in sheltered areas (e.g., under loosened tree bark) in the vicinity of fall host plants, which often include Kudzu. Adults become active again in the spring and search for suitable host plants. Adult females lay an average of 16 eggs per cluster on leaf nodes or on the undersides of leaves (Zhang et al. 2012). There are 2 generations per summer in the Southeast. Eggs are covered with symbiotic bacteria from the mother, which the offspring consume after they hatch. Juvenile insects stay together on the host plant, normally around the leaf buds (Eger et al. 2010). Gardner et al. (2013) collected Kudzu Bugs from 33 plant species representing 15 families. Approximately half of the host plants were members of the Fabaceae (legume family), and Kudzu was the most frequently reported host. Currently, Kudzu and Soybean are the only confirmed reproductive host plants of the Kudzu Bug in the North American range (Zhang et al. 2012). Our research examined seasonal dynamics of the Kudzu Bug and its resting and feeding-plant preference. We hypothesized that insect abundance at the Oconee River Greenway in Baldwin County, GA, would increase throughout the growing season and then decrease in the fall. Baldwin County is located in central Georgia along the Fall Line that separates the Piedmont and the Coastal Plain physiographic provinces. Because Kudzu Bugs have the potential to impact commercially important crops, we examined preference among 3 legume species: Soybean, Phaseolus lunatus L. (Lima Bean), and Kudzu. Soybeans are an economically important crop grown for oil production, animal protein, and human consumption. Recent research has shown that Kudzu Bug herbivory can decrease Soybean yields (Seiter et al. 2012). Lima Beans are also cultivated for human and animal consumption, and they are often used as a cover crop. Kudzu is found ubiquitously along roadsides and other habitats throughout the Southeast where it is one of the region’s most serious invasive plant species (Blaustein 2001). Additionally, we investigated if Soybean inoculation with Rhizobium spp. (rhizobium) influenced host-plant choice. We examined preference between the following pairs: Soybeans and Lima Beans, Soybeans and Kudzu, and rhizobium-inoculated and non-inoculated Soybeans. Materials and Methods Seasonal dynamics The Oconee River Greenway is a community park adjacent to the Oconee River in Milledgeville, GA (33.08°N, 83.21°W). We chose 10 sites at the Greenway within an area of 0.51 km2 based on Kudzu abundance. Sites were 3 m2, were separated by wooded areas, and contained only Kudzu. We sampled sites once a week at 16:00 hr from 3 May until 20 November 2012, except for a 2-week interval in mid-summer. We used sweep nets to collect the samples by sweeping the nets Southeastern Naturalist 59 S.M. Huskisson, K.L. Fogg, T.L. Upole, and C.B. Zehnder 2015 Vol. 14, No. 1 through the Kudzu vines 15 times at each site. We placed the collected insects in Ziplock® bags with cotton balls soaked in ethyl acetate and kept them in the freezer until they could be counted and sorted based on age-class (adults versus juveniles). We determined insect-age class by size and the presence or absence of fully developed wings. Plant-preference tests We grew Soybeans and Lima Beans in the Georgia College campus greenhouse. Seeds were planted individually in 10 cm x 10 cm plastic pots. We watered the plants as needed, they received natural light, and were regularly rotated to reduce the effects of environmental variation within the greenhouse. The seedlings were ~5 weeks old at the start of the experiments. For the Kudzu used in the preference tests, we placed branches collected from the Oconee River Greenway in water and used within 24 h. We conducted 3 sets of preference tests in the greenhouse: Soybeans vs. Lima Beans, Soybeans vs. Kudzu, and rhizobium-inoculated vs. non-inoculated Soybeans. For each test, we set up mesh cages (BugDorm 2120, MegaView Science Company, Taichung, Taiwan) that measured 60 cm x 60 cm x 60 cm and contained the plant pairs described below. We purchased the Butterbean Soybean variety from Johnny’s Selected Seeds (Albion, ME) and Burpee’s Lima Fordhook 242 Bush seeds from Lowe’s (Lowe’s, Mooresville, NC). We made visual estimates to qualitatively ensure that biomass was equal for each species in each cage. We conducted the Soybean vs. Lima bean tests in September 2012. We placed 15 cages in the greenhouse, each with one 5-week-old non-inoculated Soybean seedling and one 5-week-old Lima Bean seedling. We placed 15 adult Kudzu Bugs collected from a variety of plants including Kudzu from the Oconee River Greenway in each cage within 4 h of collection. We then monitored the insects once a day between 13:00 and 15:00 h for the following 9 days and recorded the number of insects per plant for each cage starting 24 h after the initial setup. On average, 3 insects per cage died over the course of the choice-preference test. We conducted preference tests between Soybeans and Kudzu in September 2013. We grew 40 non-inoculated Soybean seedlings in individual pots in the greenhouse. We prepared 20 cages, each containing one Soybean plant, a visually equivalent amount of Kudzu, and 10 Kudzu Bugs. We monitored each cage twice daily for 48 h after initial setup—once at ~08:00 h and again at ~16:00 h. We conducted preference tests between inoculated and non-inoculated Soybeans in October 2013. We grew 40 Soybean seedlings in individual pots in the greenhouse. We randomly selected 20 of the Soybean seedlings and inoculated them with Bradyrhizobium japonicum, a nitrogen-fixing bacterium (N-DURE A Premium Inoculant for Soybeans, Lot # K165, INTX Microbials, LLC, Kentland, IN) using the slurry method. The other 20 seedlings were non-inoculated. We set up 20 cages, each containing one non-inoculated Soybean plant, an equivalently- sized inoculated Soybean plant, and 10 Kudzu Bugs; preference tests were conducted as described above for the Kudzu–Soybean preference tests. Southeastern Naturalist S.M. Huskisson, K.L. Fogg, T.L. Upole, and C.B. Zehnder 2015 Vol. 14, No. 1 60 We treated each cage as a replicate in the preference tests. We analyzed Kudzu Bug preference using a two-tailed t-test in Microsoft Excel, with plant type as the independent variable and the number of insects as the dependent variable. These analyses only included insects present on plants and did not include insects that were elsewhere in the cage because we were only concerned with insects that were feeding or resting on the plants. Results Seasonal dynamics Our Kudzu Bug sampling at the Oconee River Greenway lasted 7 months, and included 24 sampling occurrences; in total, we collected 8860 insects. Kudzu Bug peak relative abundance occurred in early July through late August (Fig. 1). We found adults at high abundances throughout the summer, while juveniles showed a peak in early July and then another small peak in September. The relative abundance of insects in both life stages sharply declined in November. Plant preference In the preference tests between Soybeans and Lima Beans, Kudzu Bugs showed a significant preference for Soybeans over Lima Beans (Table 1). On all 9 days of the experiment, Kudzu Bug density was significantly higher on the Soybean seedlings than on the Lima Bean seedlings. In the preference tests between Kudzu and Soybeans, Kudzu was significantly preferred over Soybeans during the afternoon sampling of the first day (Day 1 AM t38 = 1.38, P = 0.180; Day 1 PM t38 = 2.34, P = 0.025; Day 2 AM t38 = 1.38, P = 0.175; Day 2 PM t38 = 1.44, P = 0.159; Fig. 2). Kudzu Bugs did not show any preference between inoculated and non-inoculated Soybeans (Day 1 AM t38 = 0.254, P = 0.801; Day1 PM t38 = 0.677, P = 0.502; Day 2 AM t38 = 0.085, P = 0.933; Day 2 PM t38 = 1.529, P = 0.135; Fig. 3). Discussion We investiagted seasonal abundance patterns and plant preferences of Kudzu Bugs. The insects preferred Soybeans over Lima Beans. When Kudzu and Soybeans were compared as food sources, Kudzu Bugs prefered Kudzu during one of Table 1. Mean Kudzu Bug density (SD) on Soybean or Lima Bean (n = 15) on each day of the feedingpreference test. Mean insect density on Soybean versus Lima Bean for each day was compared using a 2-tailed t-test (df = 28 for all t-tests). These analyses only included insects on plants and did not include insects that were on the cage itself. Day 1 Day 2 Day 3 Day 4 Day 5 Day 6 Day 7 Day 8 Day 9 Soybean 6.20 6.93 4.93 6.53 5.80 4.07 5.67 5.13 3.53 (2.3) (2.1) (1.7) (2.8) (2.5) (3.0) (3.3) (3.4) (2.7) Lima Bean 2.33 2.87 1.67 1.87 1.60 1.07 0.80 1.00 0.93 (2.9) (2.5) (1.6) (2.0) (2.2) (1.6) (1.1) (1.5) (1.6) t-stat 3.965 4.833 3.941 5.195 4.873 3.396 5.445 4.036 3.703 P-value less than 0.001 less than 0.001 less than 0.001 less than 0.001 less than 0.001 less than 0.001 less than 0.001 less than 0.001 less than 0.001 Southeastern Naturalist 61 S.M. Huskisson, K.L. Fogg, T.L. Upole, and C.B. Zehnder 2015 Vol. 14, No. 1 the observation periods. However, field trials are needed to confirm the results we obtained in the greenhouse. Our results corroborate other studies showing that during the hot summer months, when insect abundance is highest, both Soybeans and Kudzu are heavily colonized by Kudzu Bugs (Suiter et al. 2010, Zhang et al. 2012). Figure 1. Mean Kudzu Bug abundance (± standard error) per sweep of the sweep net averaged across 10 sites for (A) adults (dark gray line) and juveniles (light gray line) and (B) mean abundance for all life stages collected from the Oconee River Greenway in Milledgeville, GA, in 2012. Southeastern Naturalist S.M. Huskisson, K.L. Fogg, T.L. Upole, and C.B. Zehnder 2015 Vol. 14, No. 1 62 Our sampling at the Oconee River Greenway showed that as temperatures increased throughout the summer, there was also an increase in insect abundance (Fig. 1B). When adult abundance was at its peak, insects covered Kudzu stems and leaves (S. Huskisson and K. Fogg, Georgia College, Milledgeville, GA, pers. comm). Our results support what is currently known about the Kudzu Bug life cycle (Ruberson et al. 2013). Adults survive the winter months by overwintering in tree bark and on the ground in leaf litter (Ruberson et al. 2013). Adults emerge early in the spring in search of host plants where the insects can feed and oviposit. The first generation begins emerging in June. Adult abundance continues to increase through the summer until it peaks in late summer (Eger et al. 2010). The insects are Figure 2. Mean Kudzu Bug density (± standard error) on Kudzu and Soybean during feedingpreference test, with 10 insects added to each cage (n = 20) containing equivalent amounts of each plant material; * over a pair of bars indicates P < 0.05. Figure 3. Mean Kudzu Bug density (± standard error) on non-inoculated and inoculated Soybean plants during a feeding- preference test with 10 insects added to each cage (n = 20) containing one rhizobium-inoculated Soybean seedling and one non-inoculated Soybean seedling. Southeastern Naturalist 63 S.M. Huskisson, K.L. Fogg, T.L. Upole, and C.B. Zehnder 2015 Vol. 14, No. 1 bivoltine (Eger et al. 2010), and we observed the second generation of juveniles emerging in mid-September (Fig. 1A). Our sampling also found a large amount of variation in Kudzu Bug abundance among sites, especially during peak population densities (Fig. 1). It is possible that abiotic variation among sites—differences in temperature or humidity levels or differences in Kudzu plant quality—contributed to variation in insect density. Other insects in the order Hemiptera follow similar abundance patterns. Euschitus servus (Say) (Brown Stink Bug) exhibits seasonal dynamics in Iowa quite similar to the dynamics exhibited by Kudzu Bugs in Georgia. Brown Stink Bugs have 2 generations that are active in the summer months, and the adults overwinter (Munvaneza and McPherson 1994). Light and activity are correlated for many insect species, and many species are more active during longer photoperiods than during shorter ones (Dingle 1968). During our preference-choice tests, Kudzu Bug densities were always higher on Kudzu than on Soybeans. However, this difference was only statistically significant during the afternoon sampling period on Day 1 (Fig. 2). It is possible that the field-collected Kudzu vines were becoming less attractive to the insects because of wilting, which would explain why there was no difference on Day 2. This preference for Kudzu over Soybeans on Day 1 is interesting, given that the Kudzu samples used in this study were field-collected but the Soybeans were young seedlings growing in pots. We would have expected that the Kudzu Bugs would prefer the greenhouse-raised Soybeans over the field-collected Kudzu because fieldgrown foliage has been observed to have greater leaf toughness, a higher carbon content, higher trichome density, and lower water content than greenhouse-grown foliage (Frye et al. 2007). In this study, we were unable to differentiate Kudzu Bug feeding and resting behaviors on the plants. Additionally, in these experiments, we did not record any measure of damage by herbivory because of the short timescale and low insect densities used. It is curious that Kudzu Bugs showed no preference between inoculated and non-inoculated Soybeans. It has been reported that additional nitrogen supplied by rhizobium usually improves the food quality of plants and consequently, increases herbivore performance (Kempel et al. 2009). Other work in our laboratory has shown that inoculation causes increased root nodulation (A. Zimmerman and C.B. Zehnder, Georgia College, Milledgeville, GA, unpubl. data), providing evidence that the inoculation process worked. One field study of aphids found that Soybeans inoculated with commercially prepared rhizobium strains or given nitrogen-rich fertilizer exhibited greater insect densities compared with those that were inoculated with natural rhizobium strains (Dean et al. 2009). In this study, we did not measure plant nitrogen content, so we don’t know if nitrogen concentration differed between the inoculated and non-inoculated Soybeans. Perhaps because our plants were grown in a commercial soil mix they were not nitrogen limited, and there was no difference in nitrogen content between the inoculated and non-inoculated Soybeans. It is also interesting to note that many of the insects during this part of the experiment were found on the cage itself instead of on Soybean seedlings, especially on the first day of Southeastern Naturalist S.M. Huskisson, K.L. Fogg, T.L. Upole, and C.B. Zehnder 2015 Vol. 14, No. 1 64 the experiment. Perhaps the longer photoperiods in October caused the Kudzu Bugs to rest on the cage or begin searching for an overwintering location, rather than initiate feeding during the first day of the experiment. In summary, our study confirms previous observations of the insect’s host-plant preferences in its native Asia and in the southeastern US. Additionally, we observed that Kudzu Bugs infest Soybeans in the presence of Kudzu. Our observations of increased Kudzu Bug abundance during the summer months indicate that this insect thrives under the high temperature conditions in which Kudzu al so flourishes. Acknowledgments We thank P. Flamming, B. Patterson, A. Pierce, T. Quedensley, and A. Zimmerman for laboratory and greenhouse assistance. This research was supported by the Department of Biological and Environmental Sciences at Georgia College. Literature Cited Blaustein, R.J. 2001. Kudzu’s invasion into Southern United States life and culture. Pp. 55–62, In J.A. McNeeley (Ed.). The Great Reshuffling: Human Dimensions of Invasive Species. IUCN, Gland, Switzerland and Cambridge, UK. Dean, J.M., M.C. Mescher, and C.M. De Moraes. 2009. Plant–rhizobia mutualism influences aphid abundance on Soybean. Plant Soil 323:187–96. Dingle, H. 1968. Life history and population consequences of density, photo-period, and temperature in a migrant insect, the Milkweed Bug Oncopeltus. The American Naturalist 102:149–163. Eger, J.E., L.M. Ames, D.R. Suiter, T.M. Jenkins, and D.A. Rider. 2010. Occurrence of the Old World bug Megacopta cribraria (Fabricius) (Heteroptera: Plataspidae) in Georgia: A serious home invader and potential legume pest. Insect Mundi 121:1–11. Forseth, I.N., and A.F. Innis. 2004. Kudzu (Pueraria montana): History, physiology, and ecology combine to make a major ecosystem threat. Plant Science 23:401–413. Frye, M.J., J. Hough-Goldstein, and J.H. Sun. 2007. Biology and preliminary host-range assessment of two potential Kudzu biological control agents. Environmental Entomology 36:1430–1440. Gardner, W.A., H.B. Peeler, J. LaForest, P.M. Roberts, A.N. Sparks, Jr., J.K. Greene, D. Reisig, D.R. Suiter, J.S. Bacheler, K. Kidd, C.H. Ray, X.P. Hu, R.C. Kemerait, E.A. Scocco, J.E. Eger, J.R. Ruberson, E.J. Sikora, D.A. Herbert, C. Campana, S. Halbert, S.D. Stewart, C. Campana, S. Halbert, S.D. Stewart, G.D. Buntin, M.D. Toews, and C.T. Barderon. 2013. Confirmed distribution and occurrence of Megacopta cribraria (F.) (Hemiptera: Heteroptera: Plataspidae) in the Southeastern United States. Journal of Entomological Science 48:118–127. Kempel, A., R. Brandl, and M. Schädler. 2009. Symbiotic soil microorganisms as players in aboveground plant–herbivore interactions: The role of rhizobia. Oikos 118:634–40. Munyaneza, J., and J.E. McPherson. 1994. Comparative study of life histories, laboratory rearing, and immature stages of Euschistus servus and Euschistus variolarius (Hemiptera: Pentatomidae). The Great Lakes Entomologist 26:263–272. Ruberson, J.R., K. Takasu, G.D. Buntin, J. Eger, W.A. Gardner, J.K. Greene, T.M. Jenkins, A.W. Jones, D.M. Olson, P.M. Roberts, D.R. Suiter, and M.D. Toews. 2013. From Asian curiosity to eruptive American pest: Megacopta cribraria (Hemiptera: Plataspidae) and prospects for its biological control. Applied Entomology and Zoology 48(1):3–13. Southeastern Naturalist 65 S.M. Huskisson, K.L. Fogg, T.L. Upole, and C.B. Zehnder 2015 Vol. 14, No. 1 Seiter, N.J., J.K. Greene, and F.P.F. Reay-Jones. 2012. Reduction of Soybean yield components by Megacopta cribraria (Hemiptera: Plataspidae). Journal of Economic Entomology 106(4):1676–1683. Suiter, D.R., J.E. Eger, W.A. Gardner, R.C. Kemerait, J.N. All, P.M. Roberts, J.K. Greene, L.M. Ames, G.D. Buntin, T.M. Jenkins, and G.K. Douce 2010. Discovery and distribution of Megacopta cribraria (Hemiptera: Heteroptera: Plataspidae) in Northeast Georgia. Journal of Integrated Pest Management 1:F1–F4. Zhang, Y., J.L. Hanula, and S. Horn. 2012. The biology and preliminary host range of Megacopta cribraria (Heteroptera: Plataspidae) and its impact on Kudzu growth. Environmental Entomology 41:40–50.