ARTHROPOD BIOLOGY Isolation and DNA Barcode Characterization of a Permanent Telenomus (Hymenoptera: Platygastridae) Population in Florida That Targets Fall Armyworm (Lepidoptera: Noctuidae) MIRIAN M. HAY-ROE,1,2 RODNEY N. NAGOSHI,1,3 ROBERT L. MEAGHER,1 4 5 MYRIAM ARIAS DE LOPEZ, AND ROGELIO TRABANINO Ann. Entomol. Soc. Am. 108(5): 729–735 (2015); DOI: 10.1093/aesa/sav074 ABSTRACT Telenomus remus Nixon is a platygastrid egg parasite of the fall armyworm, Spodoptera frugiperda (J. E. Smith), with a history of use as an augmentative biological control agent in Central and South America. Efforts were made in 1975–1977 and again in 1988–1989 to introduce T. remus into the fall armyworm overwintering regions of southern Florida to mitigate infestations by this migratory pest, but in neither case was evidence of long-term establishment found. However, in 2009 and again in 2013, an unidentified Telenomus species was found attacking fall armyworm sentinel egg masses placed on corn plants or pasture grasses in the north-central Florida counties of Levy and Alachua. Taxonomic uncertainties have so far not allowed a conclusive identification of species by morphological keys. DNA barcode comparisons showed a single Florida haplotype in all collections that was identical to that found in a T. remus colony from Ecuador and very similar to a T. remus colony from Honduras. The T. remus barcode sequences were phylogenetically distinct from a second Telenomus species from Ecuador, T. rowani, and from other related sequences obtained from the NCBI GenBank database. This repre- sents the first observation of a permanent Telenomus population in the United States that targets fall armyworm and provides genetic evidence for its identification as T. remus. These findings have positive implications for the use of augmentative biological control methods to mitigate fall armyworm migration from Florida. KEY WORDS Spodoptera frugiperda, parasitoid, biological control Telenomus remus Nixon (Hymenoptera: Platygastridae) times with successful establishment demonstrated in is a lepidopteran egg parasitoid that is currently being parts of the Caribbean, Venezuela, and Honduras mass reared and released in several Central and South (reviewed in Cave 2000), and evidence found for per- American countries for the control of Spodoptera (Noc- manent populations in Ecuador (Pomari et al. 2012). tuidae) pest species, in particular the fall armyworm Augmentative releases of T. remus and other Telenomus Spodoptera frugiperda (J. E. Smith) (Cave 2000). This species continue to be used in biological control pro- small parasitic wasp is noteworthy for its aggressive and grams in Central and South America, so the geographi- efficient ability to parasitize the large egg masses asso- cal distribution of these species in this region is likely ciated with fall armyworm, whose multiple, superim- to be both variable and expanding (Figueiredo et al. posed layers are covered with scales that limit attack 2002, Van Lenteren and Bueno 2003, Gutie´rrez- from other parasitoids (Cave and Acosta 1999). Martı´nez et al. 2012). T. remus can penetrate all layers of the egg mass, rou- Cave (2000) discussed the difficulty of distinguishing tinely producing 80–100% parasitization in laboratory T. remus from native species that have overlapping host studies (Wojcik et al. 1976, Pomari et al. 2012), charac- range. Specifically, Telenomus minutissimus Ashmead is teristics that favor its use as a biological control agent. native to the Caribbean and can parasitize Spodoptera T. remus is a native of Asia and has been introduced spp., as does Telenomus solitus Johnson, a native of into the Western Hemisphere in multiple locations and Guatemala and Honduras (Yaseen et al. 1981, Cave 2000). Both species have similar external morphologies with T. remus, including the male genitalia (Cave 2000). In addition, Telenomus nawai Ashmead and Tel- 1 Center for Medical, Agricultural and Veterinary Entomology, enomus spodopterae (Dodd) are Eastern Hemisphere USDA-ARS, Gainesville, FL 32608. 2 Everglades Research and Education Center, University of Florida, species with similar host ranges as T. remus,with Belle Glade, FL 33430. T. nawai having been released in the Caribbean in 3 Corresponding author, e-mail: [email protected]. 1980 (Braithwaite and Pollard 1981). It has been sug- 4 Instituto Nacional Autonomo de Investigaciones Agropecuarias, gested that T. nawai, T. spodopterae,andT. minutissi- Sede Estacio´n Experimental Litoral del Sur, Km 26.5 Vı´a Duran – Tambo, Parroquia Virgen de Fa´tima, Yaguachi, Apartado Postal: 09 1 mus are synonymous species with T. remus, though this 7069, Guayas, Ecuador. has not been established (Braithwaite and Pollard 5 Zamorano, Apartado Postal 93, Tegucigalpa, Honduras. Published by Oxford University Press on behalf of Entomological Society of America 2015. This work is written by US Government employees and is in the public domain in the US. 730 ANNALS OF THE ENTOMOLOGICAL SOCIETY OF AMERICA Vol. 108, no. 5 1981, Yaseen et al. 1981, Centre for Agriculture Bio- the Instituto Nacional Autonomo de Investigaciones science International (CABI) 2014). These taxonomic Agropecuarias, Guayas, Ecuador. The length of time uncertainties and morphological ambiguities complicate in colony and frequency of new field infusions are attempts to identify and characterize the populations of uncertain. Fall armyworm eggs from a laboratory col- T. remus and closely related species in the Western ony were placed in rice fields where they were vulner- Hemisphere. able to infestation by parasitoids like Telenomus. Fall armyworm is the primary pest of sweet corn and Similarly, T. rowani were collected using sentinel eggs forage corn in Florida and is an important pest of corn, from a laboratory colony of the rice borer Rupela albi- sorghum, and turf grasses in the United States, with nella (Cramer) placed in rice fields. Colonies were occasional breakout years of more intensive infestations maintained on fall armyworm or R. albinella eggs with (Luginbill 1928, Sparks 1979). The pest can be found periodic supplementation with field-collected speci- throughout the central and eastern United States, mens. Telenomus remus from Honduras was obtained extending as far north as Canada, but is only able to from laboratory colonies maintained at the Escuela overwinter in southern Florida and Texas (Pair and Agricola Panamericana, Zamorano, Tegucigalpa, Sparks 1986, Nagoshi et al. 2012). It has long been Honduras. thought that controlling the overwintering populations Collecting Telenomus in Florida. Telenomus with natural enemies, such as T. remus, could be an specimens were collected in the spring of 2009 using effective means of mitigating the migratory infestations sentinel egg masses as part of a multi-year study on fall (Wojcik et al. 1976, Knipling 1980, Waddill and Whit- armyworm natural enemies in Florida. Fall armyworm comb 1982). With this objective in mind, there was an egg masses were obtained from colonies maintained at attempt in 1975–1977 to establish T. remus in the over- the USDA-ARS, CMAVE Behavior and Biocontrol wintering regions of Florida with the release of over Unit in Gainesville, FL. Females were allowed to ovi- 660,000 adults in the Homestead area just south of the posit on paper towels the night before the experiment. city of Miami (Waddill and Whitcomb 1982). These Paper segments (25 by 25 mm) containing the eggs wasps were mass reared in Trinidad and originated were pinned to the underside of leaves in pastures at a from populations established in Barbados that origi- cattle farm in Levy County, FL (Table 1). The eggs nated from Malaysia (Waddill and Whitcomb 1982, were left in the field for 36–48 h then brought to the Cave 2000). A second mass release in the same area laboratory for further rearing in 55 by 15 mm Petri was performed in 1988–1989 with approximately dishes. Emerging fall armyworm larvae were promptly 110,000 adults that originated from established popula- removed to avoid egg damage. A subset of the adult tions in Puerto Rico and the Cayman Islands (Bennett parasitoids that emerged from these field-collected 1994, Releases of Beneficial Organisms in the United eggs was preserved in 70% ethanol and stored until States and Territories [ROBO] 2008). In neither case taxonomic identification and DNA analysis. The were parasitoids recovered after termination of the remaining parasitoids were used to establish a labora- releases, indicating that the establishment of a large tory colony using the following procedure. The parasi- population was unsuccessful. toids were maintained in a clear, round, wide mouth This paper describes the existence since 2009 of a plastic containers with screw caps that were 13.5 (d) by Telenomus population in north-central Florida that 14 (h) cm. The containers were placed upside down so uses fall armyworm as a host, and provides genetic evi- that the bottom of the container was kept facing dence that it is likely to be T. remus. DNA barcoding upward. A circular 6-cm screen was placed on the bot- methods were used to better characterize the Florida toms of the containers for ventilation and the screw lid population and its relationship with Telenomus popula- was used when access to the colony was required. Sev- tions in Central and South America (Hebert et al. eral smears of honey were placed on the interior walls 2003). In this technique, the DNA sequence of a of the containers and pieces of paper towel saturated portion of the mitochondrial Cytochrome Oxidase with water were embedded in a 30-ml acrylic Solo cup Subunit I gene (COI) was compared among different (Solo Cup Co., Urbana, IL) for parasitoid feeding. The populations. The degree of sequence similarity parasitoids were allowed to mate for 3 d and then typically relates to the taxonomic relatedness of the masses of fall armyworm eggs were offered for oviposi- populations, which can be measured by phylogenetic tion. All rearing was done at 24C, 65% humidity, and analysis.