The First Record of Ooencyrtus nezarae (Hymenoptera: Encyrtidae) on Kudzu Bug (Hemiptera: Plataspidae) in North America

The First Record of Ooencyrtus nezarae (Hymenoptera: Encyrtidae) on Kudzu Bug (Hemiptera:... The kudzu bug, Megacopta cribraria (F.) (Heteroptera: Plataspidae), is an invasive insect pest introduced from Asia in 2009 that poses a threat to soybeans (Glycine max [L.] Merr. [Fabales: Fabaceae]) and other legume crops in the United States. Initially discovered in Georgia, M. cribraria rapidly expanded across the southeast until 2014 when a significant decline in its population was observed across many locations. This notable decline in M. cribraria populations is attributed to the emergence of new parasitoids and pathogens in its new invasive range. So far, only a single egg parasitoid, Paratelenomus saccharalis (Dodd) (Hymenoptera: Platygastridae), is known to parasitize the eggs of M. cribraria in the United States. Here, we report a new egg parasitoid of M. cribraria identified as Ooencyrtus nezarae Ishii, 1928 (Hymenoptera: Encyrtidae), recovered from egg masses of M. cribraria collected from soybean in Alabama. O. nezarae is reported to parasitize eggs from a variety of heteropteran families and has been observed parasitizing M. cribraria in China. To the best of our knowledge, this is the first report of O. nezarae in North America. The potentials of O. nezarae for biological control of M. cribraria in the United States and the direction of future studies are discussed. Key words: Egg parasitoid, biological control, kudzu bug, ecological guild Megacopta cribraria (F.) (Hemiptera: Plataspidae), also known as good hitchhiker (Suiter et al. 2010). M. cribraria has emerged as the kudzu bug, is an invasive insect pest accidentally introduced from top yield-limiting pest of soybean, which is the second most planted Asia to North America in 2009. Although it feeds on kudzu (Puereria field crop in the United States, having an estimated annual market montana var. lobata (Willdenow) Ohwi, (Fabales: Fabaceae) – an value of about $39 billion (USDA-ERS 2016). M. cribraria can cause economically important invasive weed native to Asia – it also feeds up to 60% yield loss in soybean (Seiter et al. 2013). In urban areas, on soybeans (Glycine max [L.] Merr. [Fabales: Fabaceae]) and other M.  cribraria is considered a nuisance pest as it invades homes to legume crops, causing significant yield loss in highly infested soy- overwinter (Suiter et  al. 2010). Apart from being a nuisance and beans (Fig. 1A and B) (Eger et al. 2010, Ruberson et al. 2013, Seiter an agricultural pest, M.  cribraria also affects international trade et al. 2013). M. cribraria and other closely related species have been and commerce. For instance, the Honduran government temporar- previously reported in many countries across the continents of Asia ily banned all agricultural imports from Alabama, Georgia, South and Australia (Eger et al. 2010). However, M.  cribraria is the only Carolina, and North Carolina due to detection of dead M. cribraria member of the family Plataspidae reported in North America (Eger adults in shipping containers (Ruberson et al. 2013). et  al. 2010, Suiter et  al. 2010). Since its first detection in 2009 in Adult M. cribraria are 4–6 mm long, oblong, and greenish-brown Georgia, the distribution of M. cribraria has rapidly expanded across in color (Fig. 1C) (Eger et al. 2010). Females lay eggs in groups of many states in the southern United States, including Alabama, South two parallel rows mostly on the undersides of leaves and apices Carolina, North Carolina, Florida, Tennessee, Mississippi, Virginia, of shoots (Fig. 1E and F). Nymphs (Fig. 1D) undergo five instars, Kentucky, Louisiana, Arizona, Maryland, and Delaware (Gardner and depending on temperature, the entire life cycle from egg to 2016). This rapid spread is attributed to its ability as strong flyer and adult takes approximately 6–8 wks (Srinivasaperumal et al. 1992, Published by Oxford University Press on behalf of Entomological Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. This Open Access article contains public sector information licensed under the Open Government Licence v2.0 (http://www.nationalarchives.gov.uk/doc/ open-government-licence/version/2/). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/8/4828345 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Journal of Insect Science, 2018, Vol. 18, No. 1 Figure  1. Megacopta cribraria. (A) M.  cribraria damage in soybean. (B) Close-up of M.  cribraria clustering on a soybean plant. (C) M.  cribraria adult. (D) M. cribararia nymph. (E) adult on egg mass. (F) detail of egg mass. Thippeswamy and Rajagopal 2005, Zhang et al. 2012, Del Pozo- Although several parasitoids and a pathogen have been reported Valdivia and Reisig 2013). On soybean, both adults and nymphs to attack M. cribraria in its native range, none was recovered in the suck sap from stems, petioles, and leaves (Stubbins et al. 2017), natural enemy surveys conducted in the United States prior to 2013 resulting in lower number of pods, reduced pod weight, and poor ( Zhang et al. 2012, Ruberson et al. 2013). Egg masses of M. cri- seed set (Seiter et al. 2013). M. cribraria completes two generations braria collected in 2010 and 2011, either monitored for parasitoid per year on both soybean and kudzu in the southeastern United emergence or exposed to native parasitoids, indicated the inability of States, although development may occur on secondary hosts such as native parasitoids in the United States to control M. cribraria (Zhang lima bean (Phaseolus lunatus L. [Fabales: Fabaceae]) and pigeon pea et al. 2012, Ruberson et al. 2013). Natural enemies of M. cribraria (Cajanus cajan L. [Fabales: Fabaceae]) (Zhang et al. 2012, Medal in its native range include the entomopathogenic fungus Beauveria et al. 2013, Ruberson et al. 2013, Seiter et al. 2013, Blount et al. bassiana (Balsamo) (Hypocreales: Clavicipitaceae) (Borah and Dutta 2015). In early spring (March–April), adults emerge from overwin- 2002), Ooencyrtus nezarae Ishi (Hymenoptera: Encyrtidae) (Takasu tering sites and move on to kudzu and other available legume host and Hirose 1985, Tayutivutikul and Yano 1990, Hirose et al. 1996, plants where they feed and reproduce. The first in-field generation of Wu et al. 2006), Encarsiella (=Dirphys) boswelli (Girault) (Polaszek adults emerge during June, some of which leave kudzu to find new and Hayat 1990), P. saccharalis [= Asolcus minor, Archiphanurus host plants such as soybean where they complete the second in-field minor and Paratelenomus minor (Johnson 1996)] (Wall 1928, generation. However, greenhouse studies have shown that first-gen- Hirose et al. 1996, Wu et al. 2006). Of these natural enemies, the eration adults can develop on soybean, mung bean (Phaseolus radia- specialist egg parasitoid P. saccharalis was considered for clas- tus (L.) R. Wilczek [Fabales: Fabaceae]) and lima bean, an indication sical biological control and under investigations at the USDA-ARS that overwintering adults can bypass kudzu to feed and reproduce National Biological Control Laboratory (Ruberson et al. 2013) until on early planted soybean (Del Pozo-Valdivia and Reisig 2013, Golec its sudden appearance under field conditions in several locations et al. 2015). When temperatures and day length decline in the fall, across Georgia and Alabama in 2013 (Gardner et al. 2013). There second-generation adults seek warmer areas such as residential are speculations that P. saccharalis was probably introduced into structures and under tree bark to overwinter (Wu et al. 2006, Zhang the United States with parasitized eggs of M. cribraria from Asia et al. 2012, Lahiri et al. 2015). In the following spring, the overwin- (Gardner et al. 2013). Subsequent surveys have recovered additional tering adults become active again and move into kudzu and other natural enemies, including the entomopathogenic fungi, Beauveria legume host plants. bassiana (Balsamo) (Seiter et al. 2013, 2014; Ruberson et al. 2013), Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/8/4828345 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 1 3 three adult parasitoids, Strongygaster triangulifera (Loew), a dip- slide-mounted in Canada balsam. An additional five females and teran (Golec et al. 2013), another dipteran, Phasia robertsonii two males were dried using HMDS and then card or point mounted (Townsend) (Ruberson et al. 2013), and a mermithid nematode (USNMENT numbers 01231470–1231471, and 01231597– (Stubbins et al. 2015). 01231601). Select slide-mounted and pinned specimens were pho- In this study, we report the detection of O. nezarae parasitizing eggs tographed using the equipment and techniques outlined in Liu and of M. cribraria in soybean crops in Alabama. O. nezarae is reported to Mottern (2017). parasitize eggs of species from several heteropteran families, includ- There is no recent, comprehensive key to the world Ooencyrtus ing Pentatomidae, Coreidae, Alydidae, and Plataspidae (see Zhang et fauna. Therefore, we used major regional keys, including those al. 2005 for a detailed host list). Notable hosts include Homoeocerus covering the Indo-Pacific region (Huang and Noyes 1994), China unipunctatus (Thunberg) [Heteroptera: Coreidae], M. punctatissimum (Zhang et  al. 2005), sub-Saharan Africa (Prinsloo 1987), and the (Montandon) (Takasu and Hirose 1985 1986), Riptortus pedestris (F.) Neotropics (Noyes 2007, 2010). We further examined new species [Heteroptera: Alydidae], Piezodorus hybneri (Gmelin) [Heteroptera: descriptions for those species described since the regional keys were Pentatomidae], Eysarcoris guttiger (Thunberg) [Heteroptera: published, and for species not included in any of the diagnostic keys. Pentatomidae], (Takasu and Hirose 1985, Hirose et al. 1996), M. cri- Specimens were also compared against the extensive Ooencyrtus braria (Wu et al. 2006), Leptocorisa chinensis Dallas [Heteroptera: collection at the U.S. National Museum of Natural History (USNM). Alydidae] (Yokosuka et al. 1998), Nezara viridula (Linnaeus) Finally, images of both slide and point-mounted specimens were sent [Heteroptera: Pentatomidae] (Jones 1988), Nezara sp., Euschistus to Dr. John Noyes at the Natural History Museum, London to fur- sp., Acrosternum sp., Edessa sp., and Thyanta sp. (Kobayashi and ther confirm our initial identification. All specimens used for species Cosenaza 1987). Until now, the distribution of O. nezarae has been determination are deposited in the USNM. limited to China, Japan, Thailand, South Korea, and Brazil (Kobayashi and Cosenza 1987, Zhang et al. 2005, Noyes 2016). To our knowl- DNA Sequencing edge, this is the first report of O. nezarae in North America and also the DNA was nondestructively extracted from one female and one first report of this species parasitizing M. cribraria in its invasive range. male specimen (USNMENT01231257 and USNMENT01231258, respectively) using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer’s protocol. Extraction, PCR ampli- Materials and Methods fication, purification, sequencing, and sequence verification/as- Collection of Parasitoid and Parasitism Rate sembly followed the protocols outlined in Liu and Mottern (2017). Parasitoids were reared from parasitized egg masses of M.  cribraria Amplified gene regions included an 815-bp fragment of the small collected from soybean fields within the Auburn University campus, subunit 18S rDNA (GenBank: KY952632 and KY952633), large Lee County, AL. (32.5934°N, 85.4952°W). Egg masses were collected subunit 28S rDNA expansion regions D2-5 (GenBank: KY965821 in the summer (July and August) of 2016. Field-collected egg masses and KY965822; performed in two reactions; see Table 1 for primer were carefully detached from soybean leaves with a pair of forceps. sequences) and a 1,041-bp fragment of cytochrome oxidase subunit Each egg mass was placed in a 2 ml Eppendorf microcentrifuge tube I  (COI) (GenBank: KY964494 and KY964495; performed in two (Eppendrof, Hauppauge, NY) and kept under laboratory conditions reactions; see Table 1 for primer sequences). All molecular work was [25 ± 1°C, 75 ± 5% RH and 14:10 (L: D) h] until emergence. Three performed at the Smithsonian Institution Laboratories of Analytical to four holes were made on the cap of each Eppendorf tube with an Biology. insect pin to allow aeration. Microscopic examination of emerged par- asitoids showed a hymenopteran with morphological features strik- Results and Discussion ingly different from P.  saccharalis. Newly emerged parasitoids were preserved in 95% ethanol and sent to the Systematic Entomology In the present study, all collected hymenopteran parasitoids from Laboratory at the National Museum of Natural History (Smithsonian M. cribraria eggs were identified as O. nezarae – an exotic egg para- Institute, Washington, DC) for identification. Voucher specimens have sitoid reported for the first time in North America. Additionally, an been deposited at the Auburn University Natural History Museum, average of 95.6% parasitism rate was recorded for O. nezarae, sug- Auburn, AL and the Smithsonian Institute, Washington, DC. gesting that this new encyrtid may serve as potential biological con- To determine field abundance and sex ratio, we sampled two soy- trol agent of M. cribraria in the United States. bean fields located in the Auburn University campus, Lee County, AL O. nezarae is a facultative gregarious egg parasitoid that attacks (32.5934°N, 85.4952°W) on five dates in July and August of 2016. In a variety of important hemipteran pests, including Homoeocerus each sampling event, egg masses were collected at random by detach- unipunctatus (Thunberg) (Hemiptera: Coreidae), Megacopta ing a whole leaf containing egg mass. Field-collected egg masses were punctatissima (Montandon) (F.) (Heteroptera: Plataspidae), and incubated individually in petri dishes (35 mm diameter) under labo- Riptortus pedestris (F.) (Hemiptera: Alydidae), in Japan (Hirose ratory conditions [25 ± 1°C, 75 ± 5% RH and 14:10 (L: D) h] and et al. 1996, Takasu et al. 2002). O. nezarae belongs to a large spe- observed for parasitoid emergence. Parasitoids that emerged were cies complex, generally characterized by a completely dark brown placed in 95% ethanol for later identification. Eggs where nothing head and body (Fig.  2A and B), dark coxae, at least partially dark emerged or hatched were dissected for signs of underdeveloped or femora and tibia, and mesoscutellar sculpture that is distinctly developed parasitoids. Number of eggs per mass, total number of par- deeper and more pronounced than the sculpture of the mesoscutum asitoids that emerged per mass, ratio of male to female (separated (Fig.  2C). The species within this group are difficult to distinguish based on antennal morphology) and percent parasitism were recorded. because the diagnostic morphological characteristics often include subtle variations in size ratios among body parts. The only diag- Parasitoid Identification nostic keys where our specimens reach a noncontradictory con- The two specimens used for gene sequencing were dried using hex- clusion are those of Huang and Noyes (1994) where they run to amethyldisilazane (HMDS) (Heraty and Hawks 1998), and then Ooencyrtus hercle Huang and Noyes (Hymenoptera: Encyrtidae), Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/8/4828345 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Journal of Insect Science, 2018, Vol. 18, No. 1 Table 1. Primers used for this study Primer Sequence Reference 18S F 5′-AAA TTA CCC ACT CCC GGC A-3′ Munro et al. (2011) 18S R 5′-TGG TGA GGT TTC CCG TGT T-3′ Munro et al. (2011) 28S D2-3551 F 5′-CGG GTT GCT TGA GAG TGC AGC-3′ Modified from Campbell et al. (2000) 28S D2-4039 R 5′-CTC CTT GGT CCG TGT TTC-3′ Mottern and Heraty (2014) 28S D3-4046 F 5′-TTG AAA CAC GGA CCA AGG AG-3′ Modified from Nunn et al. (1996) 28S D5-4625 R 5′-CGC CAG TTC TGC TTA CCA-3′ Modified from Nunn et al. (1996) COI LCO-1490 F 5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′ Folmer et al. (1994) COI HCO-2198 R 5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′ Folmer et al. (1994) COI NJ-2197 F 5′-TAT ATT TTA ATT YTW CCW GGA TTT GG-3′ Modified from Simon et al. (1994) COI MD-2614 R 5′-ATT GCA AAT ACT GCA CCT AT-3′ Dowton and Austin (1997) Numbers following Ribosomal primers refer to the complimentary 5′ start position in Drosophila melanogaster Meigen (Diptera: Drosophilidae) (Tautz et al. 1988). Numbers following cytochrome oxidase subunit I primers refer to the complimentary 5′ start position in Drosophila yakuba Burla (Diptera: Drosophilidae) (Folmer et al. 1994). The complete 28S D2-5 was amplified in two reactions: one for D2 and a second for D3-5. Figure 2. Ooencyrtus nezarae. (A) Female habitus with inset showing detail of mandible. Note the long, blade-like truncation with crenulations on the mandible, resulting in a serrated appearance. (B) Male habitus. (C) Female dorsal mesosoma. (D) Female forewing. (E) Female hindwing. (F) Female antenna. (G) Male antenna. and the key in Zhang et al. (2005), where they run to O.  nezarae. antenna (Fig. 2G). Furthermore, our specimens do not exactly match However, our specimens are a poor match with the description and the original description of O. nezarae, which states, in reference to figures of O. hercle, particularly with respect to the form of the male the female antenna, that the first two funicular segments should be Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/8/4828345 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 1 5 slightly longer than wide, whereas they are quadrate, or even slightly It is not clear whether the Ooencyrtus sp. reported attacking wider than long in our specimens (Fig.  2F) are. However, females M. cribraria eggs in Virginia (Dhammi et al. 2016) is also O. nezarae. of our specimens are smaller in overall body length (0.70–0.77mm) The male specimen figured in Dhammi et  al. (2016) appears to than the O.  nezarae holotype (0.9  mm), upon which the original have relatively broad antennal flagellomeres compared with our description is based. To address the possibility that our specimens specimens, and the coloration of the midlegs appears much lighter. are unusually small O.  nezarae that are exhibiting allometric scal- However, direct examination of the specimens, preferably com- ing of antennal segments, we sent a series of photographs to Dr. bined with molecular comparisons, would be required to determine John Noyes, an encyrtid specialist at the Natural History Museum whether or not the wasps reported from Virginia are also O. nezarae. in London (NHML). He examined a series of authoritatively iden- O. nezarae used in this study were collected from soybean be- tified O. nezarae in their collection (six females and 10 males), and tween July and August of 2016. Although O. nezarae is reported to found that smaller specimens do have relatively shorter funicular occur in soybean from May to October in China (Wu et al. 2006) segments. Apart from overall body size (females ranged from 0.88 and June to October in Japan (Takasu and Hirose 1985, 1986), the to 1.07 mm), the specimens in NHML were a good morphological precise period when O. nazarae appears in soybean fields in United match with our specimens. These data, combined with the known States is not yet known. Both O. nezarae and P. saccharalis occupy records of O.  nezarae attacking eggs of M.  cribraria in China, led the same ecological guild and have been observed together in the us to conclude that the specimens were best identified as a small field parasitizing eggs of Megacopta species in Japan and China with form of O.  nezarae. It is worthy of note that although this parasi- O. nezarae being the dominant of the two (Takasu and Hirose 1986, toid is gregarious when developing in a large host species, it behaves Wu et al. 2006). Parasitism rate ranging from 43 to 100% in Japan as solitary in eggs of smaller host species, such as M. cribraria and (Takasu and Hirose 1986) and 22.4 to 76.9% in China (Wu et al. M. punctatissimum (Takasu and Hirose 1991). 2006). In the current study, we recorded parasitism rates ranged from To the best of our knowledge, ours are the first O. nezarae nucleo- 82.8 to 100% with an average of 95.6% (table 2). Here, the para- tide sequences to be uploaded to a public database. A BLAST search sitism rate is defined as the percentage of the total number of para- on the COI barcode sequence indicates that the closest match cur- sitized eggs, including eggs from which parasitoids failed to emerge. rently in GenBank is Ooencyrtus kuvanae (Howard) (Hymenoptera: It should be noted that samples of parasitized eggs were collected Encyrtidae) (GenBank: KX868569.1), with 91% sequence iden- from a single study site and that the high parasitization rate observed tity over 99% query coverage. BLAST searches on our ribosomal may not be the same across different locations. In this study, we genes confirm that our specimens are Ooencyrtus, but do not closely observed a sex ratio of 1 male to 2.5 females. A female-biased pro- match with any sequences currently in the database, suggesting that geny of one male to three females has been reported for O. nezarae GenBank is currently not yet populated with enough Ooencyrtus se- on R. pedestris in laboratory-reared specimens (Aung et al. 2011a, quence data to help with species identification in this case. 2011b). Similarly, Hirose et al. (1996) reported a female-biased sex ratio of 1:2.4 for O. nezarae adults in a field study. Parasitism by O. nezarae can be distinguished from parasitism by P. saccharalis based Table  2. Percent parasitism and sex ratio of O.  nezarae that on the position of the emergence holes on the M. cribraria eggs: O. emerged from field-collected egg masses of M.  cribraria from a single soybean field in summer 2016 nezarae adults emerge from lateral, irregularly shaped openings (Fig. 3A), whereas P. saccharalis adults emerge from circular openings at # Eggs/Mass % Parasitism # Emerged #Male #Female the apices of the eggs (Fig. 3B). O.  nezarae has a broad geographic range. It has been reported 19 100 17 08 11 in Asia (China, South Korea, Japan, Thailand) and Brazil, where it 08 100 08 02 06 30 100 30 06 24 was introduced to control stink bugs in soybean (Zhang et al. 2005). 12 83.3 10 03 07 This range, combined with the morphological variation that we are 16 100 16 04 12 reporting, suggests the need for additional taxonomic work (inte- 15 100 15 05 10 grating molecules and morphology) in investigating the possibility 18 100 18 03 15 that O. nezarae is a cryptic species complex. 20 90 18 05 13 The discovery of O.  nezarae in Alabama under field conditions 26 100 26 09 17 is not the first egg parasitoid found attacking M.  cribraria in the 29 82.8 24 08 16 United States; P.  saccharalis was found in Georgia and Alabama Figure 3. M. cribraria egg mass showing emergence holes from O. nezarae (A). Wasps emerge from ragged, irregularly shaped holes in the sides of the eggs. M. cribraria egg mass with P. saccharalis emerging from a circular hole at the apex of the egg (B). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/8/4828345 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6 Journal of Insect Science, 2018, Vol. 18, No. 1 in 2013 (Gardner et  al. 2013). As with P.  saccharilis, the route of Dowton, M., and A. D.  Austin. 1997. Evidence for AT-transversion bias in wasp (Hymenoptera: Symphyta) mitochondrial genes and its implications introduction of O.  nezarae to North America is unknown, and we for the origin of parasitism. J. Mol. Evol. 44: 398–405. provide two possible explanations. First, we speculate that it came Eger, J. E. Jr., L. M.  Ames, D. R.  Suiter, T. M.  Jenkins, D. A.  Rider, and into the United States with parasitized eggs of M.  cribraria from S. E. Halbert. 2010. Occurrence of the Old World bug Megacopta cribraria Asia. Another possibility is that because O. nezarae is a generalist (Fabricius) (Heteroptera: Plataspidae) in Georgia: a serious home invader parasitoid, it came in with parasitized eggs of other host insects and and potential legume pest. Insecta Mundi 0121: 1–11. switched to M. cribraria due to availability, proximity, or preference. Folmer, O., M. Black, W. Hoeh, R. Lutz, and R. Vrijenhoek. 1994. DNA prim- In conclusion, two egg parasitoids, O. nezarae and P. saccharalis, ers for amplification of mitochondrial cytochrome c oxidase subunit I from have been identified for the control of M. cribraria in soybean in the diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3: 294–299. United States with parasitism rates ranging from 82.8 to 100% and Gardner, W. A., J. L. Blount, J. R. Golec, W. A. Jones, X. P. Hu, E. J. Talamas, 42 to 95%, respectively, at their reported study sites. Other parasi- R. M.  Evans, X.  Dong, C. H.  Ray Jr., G. D.  Buntin, N. M.  Gerardo, and J.  Couret. 2013. Discovery of Paratelenomous saccharalis (Dodd) toids that have been recovered include Strongygaster triangulifera (Hymenoptera: Platygastridae), an egg parasitoid of Megacopta cri- (Loew) (Diptera: Tachinidae) (Golec et  al. 2013), Phasia robertso- braria F. (Hemiptera: Plataspidae) in its expanded North American range. nii (Townsend) (Diptera: Tachinidae) (Ruberson et  al. 2013), and J. Entomol. Sci. 48: 355–359. a mermithid nematode (Stubbins et al. 2015). The mean parasitism Gardner, W. A. 2016. Megacopta cribraria distribution map. www.kudzubug. rate was 5.14% for S.  triangulifera and 4.7% for the nematode org/distribution_map.cfm (Accessed 4 April 2017). although P.  robertsonii was observed in a single adult. O.  nezarae Golec, J. R., X. P.  Hu, C. H.  Ray, and N. E.  Woodley. 2013. Strongygaster and P. saccharalis are successful parasitoids of M. cribraria both in triangulifera (Diptera: Tachinidae) as a parasitoid of adults of the invasive their native and new invasive range. Although the parasitism rate Megacopta cribraria (Heteroptera: Plataspidae) in Alabama. J. Entomol. is high for both parasitoids, they have a short period of activity in Sci 48: 352–354. the United States. With the high rate of parasitism and dominance Golec, J. R., Hu X. P., Yang L., and J. E. Eger 2015. Kudzu-deprived first-genera- tion Megacopta cribraria (F.) (Heteroptera: Plataspidae) are capable of devel- reported for O. nezarae, augmentative or inundative releases could oping on alternative legume species. J. Agric. Urban Entomol. 31: 52–61. be considered to enhance its effectiveness as a biological control Heraty, J. and D. Hawks. 1998. Hexamethyldisilazane—a chemical alternative agent of M.  cribraria in soybean production in the United States. for drying insects. Entomol. 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The First Record of Ooencyrtus nezarae (Hymenoptera: Encyrtidae) on Kudzu Bug (Hemiptera: Plataspidae) in North America

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Abstract

The kudzu bug, Megacopta cribraria (F.) (Heteroptera: Plataspidae), is an invasive insect pest introduced from Asia in 2009 that poses a threat to soybeans (Glycine max [L.] Merr. [Fabales: Fabaceae]) and other legume crops in the United States. Initially discovered in Georgia, M. cribraria rapidly expanded across the southeast until 2014 when a significant decline in its population was observed across many locations. This notable decline in M. cribraria populations is attributed to the emergence of new parasitoids and pathogens in its new invasive range. So far, only a single egg parasitoid, Paratelenomus saccharalis (Dodd) (Hymenoptera: Platygastridae), is known to parasitize the eggs of M. cribraria in the United States. Here, we report a new egg parasitoid of M. cribraria identified as Ooencyrtus nezarae Ishii, 1928 (Hymenoptera: Encyrtidae), recovered from egg masses of M. cribraria collected from soybean in Alabama. O. nezarae is reported to parasitize eggs from a variety of heteropteran families and has been observed parasitizing M. cribraria in China. To the best of our knowledge, this is the first report of O. nezarae in North America. The potentials of O. nezarae for biological control of M. cribraria in the United States and the direction of future studies are discussed. Key words: Egg parasitoid, biological control, kudzu bug, ecological guild Megacopta cribraria (F.) (Hemiptera: Plataspidae), also known as good hitchhiker (Suiter et al. 2010). M. cribraria has emerged as the kudzu bug, is an invasive insect pest accidentally introduced from top yield-limiting pest of soybean, which is the second most planted Asia to North America in 2009. Although it feeds on kudzu (Puereria field crop in the United States, having an estimated annual market montana var. lobata (Willdenow) Ohwi, (Fabales: Fabaceae) – an value of about $39 billion (USDA-ERS 2016). M. cribraria can cause economically important invasive weed native to Asia – it also feeds up to 60% yield loss in soybean (Seiter et al. 2013). In urban areas, on soybeans (Glycine max [L.] Merr. [Fabales: Fabaceae]) and other M.  cribraria is considered a nuisance pest as it invades homes to legume crops, causing significant yield loss in highly infested soy- overwinter (Suiter et  al. 2010). Apart from being a nuisance and beans (Fig. 1A and B) (Eger et al. 2010, Ruberson et al. 2013, Seiter an agricultural pest, M.  cribraria also affects international trade et al. 2013). M. cribraria and other closely related species have been and commerce. For instance, the Honduran government temporar- previously reported in many countries across the continents of Asia ily banned all agricultural imports from Alabama, Georgia, South and Australia (Eger et al. 2010). However, M.  cribraria is the only Carolina, and North Carolina due to detection of dead M. cribraria member of the family Plataspidae reported in North America (Eger adults in shipping containers (Ruberson et al. 2013). et  al. 2010, Suiter et  al. 2010). Since its first detection in 2009 in Adult M. cribraria are 4–6 mm long, oblong, and greenish-brown Georgia, the distribution of M. cribraria has rapidly expanded across in color (Fig. 1C) (Eger et al. 2010). Females lay eggs in groups of many states in the southern United States, including Alabama, South two parallel rows mostly on the undersides of leaves and apices Carolina, North Carolina, Florida, Tennessee, Mississippi, Virginia, of shoots (Fig. 1E and F). Nymphs (Fig. 1D) undergo five instars, Kentucky, Louisiana, Arizona, Maryland, and Delaware (Gardner and depending on temperature, the entire life cycle from egg to 2016). This rapid spread is attributed to its ability as strong flyer and adult takes approximately 6–8 wks (Srinivasaperumal et al. 1992, Published by Oxford University Press on behalf of Entomological Society of America 2018. This work is written by (a) US Government employee(s) and is in the public domain in the US. This Open Access article contains public sector information licensed under the Open Government Licence v2.0 (http://www.nationalarchives.gov.uk/doc/ open-government-licence/version/2/). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/8/4828345 by Ed 'DeepDyve' Gillespie user on 16 March 2018 2 Journal of Insect Science, 2018, Vol. 18, No. 1 Figure  1. Megacopta cribraria. (A) M.  cribraria damage in soybean. (B) Close-up of M.  cribraria clustering on a soybean plant. (C) M.  cribraria adult. (D) M. cribararia nymph. (E) adult on egg mass. (F) detail of egg mass. Thippeswamy and Rajagopal 2005, Zhang et al. 2012, Del Pozo- Although several parasitoids and a pathogen have been reported Valdivia and Reisig 2013). On soybean, both adults and nymphs to attack M. cribraria in its native range, none was recovered in the suck sap from stems, petioles, and leaves (Stubbins et al. 2017), natural enemy surveys conducted in the United States prior to 2013 resulting in lower number of pods, reduced pod weight, and poor ( Zhang et al. 2012, Ruberson et al. 2013). Egg masses of M. cri- seed set (Seiter et al. 2013). M. cribraria completes two generations braria collected in 2010 and 2011, either monitored for parasitoid per year on both soybean and kudzu in the southeastern United emergence or exposed to native parasitoids, indicated the inability of States, although development may occur on secondary hosts such as native parasitoids in the United States to control M. cribraria (Zhang lima bean (Phaseolus lunatus L. [Fabales: Fabaceae]) and pigeon pea et al. 2012, Ruberson et al. 2013). Natural enemies of M. cribraria (Cajanus cajan L. [Fabales: Fabaceae]) (Zhang et al. 2012, Medal in its native range include the entomopathogenic fungus Beauveria et al. 2013, Ruberson et al. 2013, Seiter et al. 2013, Blount et al. bassiana (Balsamo) (Hypocreales: Clavicipitaceae) (Borah and Dutta 2015). In early spring (March–April), adults emerge from overwin- 2002), Ooencyrtus nezarae Ishi (Hymenoptera: Encyrtidae) (Takasu tering sites and move on to kudzu and other available legume host and Hirose 1985, Tayutivutikul and Yano 1990, Hirose et al. 1996, plants where they feed and reproduce. The first in-field generation of Wu et al. 2006), Encarsiella (=Dirphys) boswelli (Girault) (Polaszek adults emerge during June, some of which leave kudzu to find new and Hayat 1990), P. saccharalis [= Asolcus minor, Archiphanurus host plants such as soybean where they complete the second in-field minor and Paratelenomus minor (Johnson 1996)] (Wall 1928, generation. However, greenhouse studies have shown that first-gen- Hirose et al. 1996, Wu et al. 2006). Of these natural enemies, the eration adults can develop on soybean, mung bean (Phaseolus radia- specialist egg parasitoid P. saccharalis was considered for clas- tus (L.) R. Wilczek [Fabales: Fabaceae]) and lima bean, an indication sical biological control and under investigations at the USDA-ARS that overwintering adults can bypass kudzu to feed and reproduce National Biological Control Laboratory (Ruberson et al. 2013) until on early planted soybean (Del Pozo-Valdivia and Reisig 2013, Golec its sudden appearance under field conditions in several locations et al. 2015). When temperatures and day length decline in the fall, across Georgia and Alabama in 2013 (Gardner et al. 2013). There second-generation adults seek warmer areas such as residential are speculations that P. saccharalis was probably introduced into structures and under tree bark to overwinter (Wu et al. 2006, Zhang the United States with parasitized eggs of M. cribraria from Asia et al. 2012, Lahiri et al. 2015). In the following spring, the overwin- (Gardner et al. 2013). Subsequent surveys have recovered additional tering adults become active again and move into kudzu and other natural enemies, including the entomopathogenic fungi, Beauveria legume host plants. bassiana (Balsamo) (Seiter et al. 2013, 2014; Ruberson et al. 2013), Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/8/4828345 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 1 3 three adult parasitoids, Strongygaster triangulifera (Loew), a dip- slide-mounted in Canada balsam. An additional five females and teran (Golec et al. 2013), another dipteran, Phasia robertsonii two males were dried using HMDS and then card or point mounted (Townsend) (Ruberson et al. 2013), and a mermithid nematode (USNMENT numbers 01231470–1231471, and 01231597– (Stubbins et al. 2015). 01231601). Select slide-mounted and pinned specimens were pho- In this study, we report the detection of O. nezarae parasitizing eggs tographed using the equipment and techniques outlined in Liu and of M. cribraria in soybean crops in Alabama. O. nezarae is reported to Mottern (2017). parasitize eggs of species from several heteropteran families, includ- There is no recent, comprehensive key to the world Ooencyrtus ing Pentatomidae, Coreidae, Alydidae, and Plataspidae (see Zhang et fauna. Therefore, we used major regional keys, including those al. 2005 for a detailed host list). Notable hosts include Homoeocerus covering the Indo-Pacific region (Huang and Noyes 1994), China unipunctatus (Thunberg) [Heteroptera: Coreidae], M. punctatissimum (Zhang et  al. 2005), sub-Saharan Africa (Prinsloo 1987), and the (Montandon) (Takasu and Hirose 1985 1986), Riptortus pedestris (F.) Neotropics (Noyes 2007, 2010). We further examined new species [Heteroptera: Alydidae], Piezodorus hybneri (Gmelin) [Heteroptera: descriptions for those species described since the regional keys were Pentatomidae], Eysarcoris guttiger (Thunberg) [Heteroptera: published, and for species not included in any of the diagnostic keys. Pentatomidae], (Takasu and Hirose 1985, Hirose et al. 1996), M. cri- Specimens were also compared against the extensive Ooencyrtus braria (Wu et al. 2006), Leptocorisa chinensis Dallas [Heteroptera: collection at the U.S. National Museum of Natural History (USNM). Alydidae] (Yokosuka et al. 1998), Nezara viridula (Linnaeus) Finally, images of both slide and point-mounted specimens were sent [Heteroptera: Pentatomidae] (Jones 1988), Nezara sp., Euschistus to Dr. John Noyes at the Natural History Museum, London to fur- sp., Acrosternum sp., Edessa sp., and Thyanta sp. (Kobayashi and ther confirm our initial identification. All specimens used for species Cosenaza 1987). Until now, the distribution of O. nezarae has been determination are deposited in the USNM. limited to China, Japan, Thailand, South Korea, and Brazil (Kobayashi and Cosenza 1987, Zhang et al. 2005, Noyes 2016). To our knowl- DNA Sequencing edge, this is the first report of O. nezarae in North America and also the DNA was nondestructively extracted from one female and one first report of this species parasitizing M. cribraria in its invasive range. male specimen (USNMENT01231257 and USNMENT01231258, respectively) using the DNeasy Blood and Tissue Kit (Qiagen) following the manufacturer’s protocol. Extraction, PCR ampli- Materials and Methods fication, purification, sequencing, and sequence verification/as- Collection of Parasitoid and Parasitism Rate sembly followed the protocols outlined in Liu and Mottern (2017). Parasitoids were reared from parasitized egg masses of M.  cribraria Amplified gene regions included an 815-bp fragment of the small collected from soybean fields within the Auburn University campus, subunit 18S rDNA (GenBank: KY952632 and KY952633), large Lee County, AL. (32.5934°N, 85.4952°W). Egg masses were collected subunit 28S rDNA expansion regions D2-5 (GenBank: KY965821 in the summer (July and August) of 2016. Field-collected egg masses and KY965822; performed in two reactions; see Table 1 for primer were carefully detached from soybean leaves with a pair of forceps. sequences) and a 1,041-bp fragment of cytochrome oxidase subunit Each egg mass was placed in a 2 ml Eppendorf microcentrifuge tube I  (COI) (GenBank: KY964494 and KY964495; performed in two (Eppendrof, Hauppauge, NY) and kept under laboratory conditions reactions; see Table 1 for primer sequences). All molecular work was [25 ± 1°C, 75 ± 5% RH and 14:10 (L: D) h] until emergence. Three performed at the Smithsonian Institution Laboratories of Analytical to four holes were made on the cap of each Eppendorf tube with an Biology. insect pin to allow aeration. Microscopic examination of emerged par- asitoids showed a hymenopteran with morphological features strik- Results and Discussion ingly different from P.  saccharalis. Newly emerged parasitoids were preserved in 95% ethanol and sent to the Systematic Entomology In the present study, all collected hymenopteran parasitoids from Laboratory at the National Museum of Natural History (Smithsonian M. cribraria eggs were identified as O. nezarae – an exotic egg para- Institute, Washington, DC) for identification. Voucher specimens have sitoid reported for the first time in North America. Additionally, an been deposited at the Auburn University Natural History Museum, average of 95.6% parasitism rate was recorded for O. nezarae, sug- Auburn, AL and the Smithsonian Institute, Washington, DC. gesting that this new encyrtid may serve as potential biological con- To determine field abundance and sex ratio, we sampled two soy- trol agent of M. cribraria in the United States. bean fields located in the Auburn University campus, Lee County, AL O. nezarae is a facultative gregarious egg parasitoid that attacks (32.5934°N, 85.4952°W) on five dates in July and August of 2016. In a variety of important hemipteran pests, including Homoeocerus each sampling event, egg masses were collected at random by detach- unipunctatus (Thunberg) (Hemiptera: Coreidae), Megacopta ing a whole leaf containing egg mass. Field-collected egg masses were punctatissima (Montandon) (F.) (Heteroptera: Plataspidae), and incubated individually in petri dishes (35 mm diameter) under labo- Riptortus pedestris (F.) (Hemiptera: Alydidae), in Japan (Hirose ratory conditions [25 ± 1°C, 75 ± 5% RH and 14:10 (L: D) h] and et al. 1996, Takasu et al. 2002). O. nezarae belongs to a large spe- observed for parasitoid emergence. Parasitoids that emerged were cies complex, generally characterized by a completely dark brown placed in 95% ethanol for later identification. Eggs where nothing head and body (Fig.  2A and B), dark coxae, at least partially dark emerged or hatched were dissected for signs of underdeveloped or femora and tibia, and mesoscutellar sculpture that is distinctly developed parasitoids. Number of eggs per mass, total number of par- deeper and more pronounced than the sculpture of the mesoscutum asitoids that emerged per mass, ratio of male to female (separated (Fig.  2C). The species within this group are difficult to distinguish based on antennal morphology) and percent parasitism were recorded. because the diagnostic morphological characteristics often include subtle variations in size ratios among body parts. The only diag- Parasitoid Identification nostic keys where our specimens reach a noncontradictory con- The two specimens used for gene sequencing were dried using hex- clusion are those of Huang and Noyes (1994) where they run to amethyldisilazane (HMDS) (Heraty and Hawks 1998), and then Ooencyrtus hercle Huang and Noyes (Hymenoptera: Encyrtidae), Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/8/4828345 by Ed 'DeepDyve' Gillespie user on 16 March 2018 4 Journal of Insect Science, 2018, Vol. 18, No. 1 Table 1. Primers used for this study Primer Sequence Reference 18S F 5′-AAA TTA CCC ACT CCC GGC A-3′ Munro et al. (2011) 18S R 5′-TGG TGA GGT TTC CCG TGT T-3′ Munro et al. (2011) 28S D2-3551 F 5′-CGG GTT GCT TGA GAG TGC AGC-3′ Modified from Campbell et al. (2000) 28S D2-4039 R 5′-CTC CTT GGT CCG TGT TTC-3′ Mottern and Heraty (2014) 28S D3-4046 F 5′-TTG AAA CAC GGA CCA AGG AG-3′ Modified from Nunn et al. (1996) 28S D5-4625 R 5′-CGC CAG TTC TGC TTA CCA-3′ Modified from Nunn et al. (1996) COI LCO-1490 F 5′-GGT CAA CAA ATC ATA AAG ATA TTG G-3′ Folmer et al. (1994) COI HCO-2198 R 5′-TAA ACT TCA GGG TGA CCA AAA AAT CA-3′ Folmer et al. (1994) COI NJ-2197 F 5′-TAT ATT TTA ATT YTW CCW GGA TTT GG-3′ Modified from Simon et al. (1994) COI MD-2614 R 5′-ATT GCA AAT ACT GCA CCT AT-3′ Dowton and Austin (1997) Numbers following Ribosomal primers refer to the complimentary 5′ start position in Drosophila melanogaster Meigen (Diptera: Drosophilidae) (Tautz et al. 1988). Numbers following cytochrome oxidase subunit I primers refer to the complimentary 5′ start position in Drosophila yakuba Burla (Diptera: Drosophilidae) (Folmer et al. 1994). The complete 28S D2-5 was amplified in two reactions: one for D2 and a second for D3-5. Figure 2. Ooencyrtus nezarae. (A) Female habitus with inset showing detail of mandible. Note the long, blade-like truncation with crenulations on the mandible, resulting in a serrated appearance. (B) Male habitus. (C) Female dorsal mesosoma. (D) Female forewing. (E) Female hindwing. (F) Female antenna. (G) Male antenna. and the key in Zhang et al. (2005), where they run to O.  nezarae. antenna (Fig. 2G). Furthermore, our specimens do not exactly match However, our specimens are a poor match with the description and the original description of O. nezarae, which states, in reference to figures of O. hercle, particularly with respect to the form of the male the female antenna, that the first two funicular segments should be Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/8/4828345 by Ed 'DeepDyve' Gillespie user on 16 March 2018 Journal of Insect Science, 2018, Vol. 18, No. 1 5 slightly longer than wide, whereas they are quadrate, or even slightly It is not clear whether the Ooencyrtus sp. reported attacking wider than long in our specimens (Fig.  2F) are. However, females M. cribraria eggs in Virginia (Dhammi et al. 2016) is also O. nezarae. of our specimens are smaller in overall body length (0.70–0.77mm) The male specimen figured in Dhammi et  al. (2016) appears to than the O.  nezarae holotype (0.9  mm), upon which the original have relatively broad antennal flagellomeres compared with our description is based. To address the possibility that our specimens specimens, and the coloration of the midlegs appears much lighter. are unusually small O.  nezarae that are exhibiting allometric scal- However, direct examination of the specimens, preferably com- ing of antennal segments, we sent a series of photographs to Dr. bined with molecular comparisons, would be required to determine John Noyes, an encyrtid specialist at the Natural History Museum whether or not the wasps reported from Virginia are also O. nezarae. in London (NHML). He examined a series of authoritatively iden- O. nezarae used in this study were collected from soybean be- tified O. nezarae in their collection (six females and 10 males), and tween July and August of 2016. Although O. nezarae is reported to found that smaller specimens do have relatively shorter funicular occur in soybean from May to October in China (Wu et al. 2006) segments. Apart from overall body size (females ranged from 0.88 and June to October in Japan (Takasu and Hirose 1985, 1986), the to 1.07 mm), the specimens in NHML were a good morphological precise period when O. nazarae appears in soybean fields in United match with our specimens. These data, combined with the known States is not yet known. Both O. nezarae and P. saccharalis occupy records of O.  nezarae attacking eggs of M.  cribraria in China, led the same ecological guild and have been observed together in the us to conclude that the specimens were best identified as a small field parasitizing eggs of Megacopta species in Japan and China with form of O.  nezarae. It is worthy of note that although this parasi- O. nezarae being the dominant of the two (Takasu and Hirose 1986, toid is gregarious when developing in a large host species, it behaves Wu et al. 2006). Parasitism rate ranging from 43 to 100% in Japan as solitary in eggs of smaller host species, such as M. cribraria and (Takasu and Hirose 1986) and 22.4 to 76.9% in China (Wu et al. M. punctatissimum (Takasu and Hirose 1991). 2006). In the current study, we recorded parasitism rates ranged from To the best of our knowledge, ours are the first O. nezarae nucleo- 82.8 to 100% with an average of 95.6% (table 2). Here, the para- tide sequences to be uploaded to a public database. A BLAST search sitism rate is defined as the percentage of the total number of para- on the COI barcode sequence indicates that the closest match cur- sitized eggs, including eggs from which parasitoids failed to emerge. rently in GenBank is Ooencyrtus kuvanae (Howard) (Hymenoptera: It should be noted that samples of parasitized eggs were collected Encyrtidae) (GenBank: KX868569.1), with 91% sequence iden- from a single study site and that the high parasitization rate observed tity over 99% query coverage. BLAST searches on our ribosomal may not be the same across different locations. In this study, we genes confirm that our specimens are Ooencyrtus, but do not closely observed a sex ratio of 1 male to 2.5 females. A female-biased pro- match with any sequences currently in the database, suggesting that geny of one male to three females has been reported for O. nezarae GenBank is currently not yet populated with enough Ooencyrtus se- on R. pedestris in laboratory-reared specimens (Aung et al. 2011a, quence data to help with species identification in this case. 2011b). Similarly, Hirose et al. (1996) reported a female-biased sex ratio of 1:2.4 for O. nezarae adults in a field study. Parasitism by O. nezarae can be distinguished from parasitism by P. saccharalis based Table  2. Percent parasitism and sex ratio of O.  nezarae that on the position of the emergence holes on the M. cribraria eggs: O. emerged from field-collected egg masses of M.  cribraria from a single soybean field in summer 2016 nezarae adults emerge from lateral, irregularly shaped openings (Fig. 3A), whereas P. saccharalis adults emerge from circular openings at # Eggs/Mass % Parasitism # Emerged #Male #Female the apices of the eggs (Fig. 3B). O.  nezarae has a broad geographic range. It has been reported 19 100 17 08 11 in Asia (China, South Korea, Japan, Thailand) and Brazil, where it 08 100 08 02 06 30 100 30 06 24 was introduced to control stink bugs in soybean (Zhang et al. 2005). 12 83.3 10 03 07 This range, combined with the morphological variation that we are 16 100 16 04 12 reporting, suggests the need for additional taxonomic work (inte- 15 100 15 05 10 grating molecules and morphology) in investigating the possibility 18 100 18 03 15 that O. nezarae is a cryptic species complex. 20 90 18 05 13 The discovery of O.  nezarae in Alabama under field conditions 26 100 26 09 17 is not the first egg parasitoid found attacking M.  cribraria in the 29 82.8 24 08 16 United States; P.  saccharalis was found in Georgia and Alabama Figure 3. M. cribraria egg mass showing emergence holes from O. nezarae (A). Wasps emerge from ragged, irregularly shaped holes in the sides of the eggs. M. cribraria egg mass with P. saccharalis emerging from a circular hole at the apex of the egg (B). Downloaded from https://academic.oup.com/jinsectscience/article-abstract/18/1/8/4828345 by Ed 'DeepDyve' Gillespie user on 16 March 2018 6 Journal of Insect Science, 2018, Vol. 18, No. 1 in 2013 (Gardner et  al. 2013). As with P.  saccharilis, the route of Dowton, M., and A. D.  Austin. 1997. Evidence for AT-transversion bias in wasp (Hymenoptera: Symphyta) mitochondrial genes and its implications introduction of O.  nezarae to North America is unknown, and we for the origin of parasitism. J. Mol. Evol. 44: 398–405. provide two possible explanations. First, we speculate that it came Eger, J. E. Jr., L. M.  Ames, D. R.  Suiter, T. M.  Jenkins, D. A.  Rider, and into the United States with parasitized eggs of M.  cribraria from S. E. Halbert. 2010. Occurrence of the Old World bug Megacopta cribraria Asia. Another possibility is that because O. nezarae is a generalist (Fabricius) (Heteroptera: Plataspidae) in Georgia: a serious home invader parasitoid, it came in with parasitized eggs of other host insects and and potential legume pest. Insecta Mundi 0121: 1–11. switched to M. cribraria due to availability, proximity, or preference. Folmer, O., M. Black, W. Hoeh, R. Lutz, and R. Vrijenhoek. 1994. DNA prim- In conclusion, two egg parasitoids, O. nezarae and P. saccharalis, ers for amplification of mitochondrial cytochrome c oxidase subunit I from have been identified for the control of M. cribraria in soybean in the diverse metazoan invertebrates. Mol. Mar. Biol. Biotechnol. 3: 294–299. United States with parasitism rates ranging from 82.8 to 100% and Gardner, W. A., J. L. Blount, J. R. Golec, W. A. Jones, X. P. Hu, E. J. Talamas, 42 to 95%, respectively, at their reported study sites. Other parasi- R. M.  Evans, X.  Dong, C. H.  Ray Jr., G. D.  Buntin, N. M.  Gerardo, and J.  Couret. 2013. Discovery of Paratelenomous saccharalis (Dodd) toids that have been recovered include Strongygaster triangulifera (Hymenoptera: Platygastridae), an egg parasitoid of Megacopta cri- (Loew) (Diptera: Tachinidae) (Golec et  al. 2013), Phasia robertso- braria F. (Hemiptera: Plataspidae) in its expanded North American range. nii (Townsend) (Diptera: Tachinidae) (Ruberson et  al. 2013), and J. Entomol. Sci. 48: 355–359. a mermithid nematode (Stubbins et al. 2015). The mean parasitism Gardner, W. A. 2016. Megacopta cribraria distribution map. www.kudzubug. rate was 5.14% for S.  triangulifera and 4.7% for the nematode org/distribution_map.cfm (Accessed 4 April 2017). although P.  robertsonii was observed in a single adult. O.  nezarae Golec, J. R., X. P.  Hu, C. H.  Ray, and N. E.  Woodley. 2013. Strongygaster and P. saccharalis are successful parasitoids of M. cribraria both in triangulifera (Diptera: Tachinidae) as a parasitoid of adults of the invasive their native and new invasive range. Although the parasitism rate Megacopta cribraria (Heteroptera: Plataspidae) in Alabama. J. Entomol. is high for both parasitoids, they have a short period of activity in Sci 48: 352–354. the United States. With the high rate of parasitism and dominance Golec, J. R., Hu X. P., Yang L., and J. E. Eger 2015. Kudzu-deprived first-genera- tion Megacopta cribraria (F.) (Heteroptera: Plataspidae) are capable of devel- reported for O. nezarae, augmentative or inundative releases could oping on alternative legume species. J. Agric. Urban Entomol. 31: 52–61. be considered to enhance its effectiveness as a biological control Heraty, J. and D. Hawks. 1998. Hexamethyldisilazane—a chemical alternative agent of M.  cribraria in soybean production in the United States. for drying insects. Entomol. News 109: 369–374. Monitoring is on-going in Alabama as well as other localities within Hirose, Y., K. Takasu, and M. Takagi. 1996. Egg parasitoids of phytophagous M. cribraria invasive range for the presence of this wasp. bugs in soybean: mobile natural enemies as naturally occurring biological control agents of mobile pests. Biol. Control 7: 84–94. Huang, D. W., and J. S.  Noyes. 1994. A revision of the Indo-Pacific species Acknowledgments of Ooencyrtus (Hymenoptera: Encyrtidae), parasitoids of the immature This study was supported by Agriculture and Food Research Initiative stages of economically important insect species (mainly Hemiptera and Competitive Grant 2015-69004-23420 from the USDA National Institute of Lepidoptera). Bull. Br. Mus. (Nat. Hist.) Entomol. 63: 1–136. Food and Agriculture and by the Alabama Agricultural Experiment Station. Johnson, N. F. 1996. 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